ANTIBODIES DIRECTED AGAINST PYROGLUTAMATE MONOCYTE CHEMOATTRACTANT PROTEIN-1 (MCP-1 N1pE)

ABSTRACT

Monoclonal antibodies which bind specifically to the proinflammatory cytokine pyroglutamate MCP-1 (MCP-1 N1pE) are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 61/090,264 filed on Aug. 20, 2008, which is incorporated herein byreference in its entirety to the extent permitted by law.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The Sequence Listing, which is a part of the present disclosure,includes a computer readable form comprising nucleotide and/or aminoacid sequences of the present invention. The subject matter of theSequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to monoclonal antibodies which bindspecifically to the proinflammatory cytokine pyroglutamate MCP-1 (MCP-1N1pE).

BACKGROUND OF THE INVENTION

Chemotactic cytokines (chemokines) are proteins that attract andactivate leukocytes and are thought to play a fundamental role ininflammation. Chemokines are divided into four groups categorized by theappearance of N-terminal cysteine residues (“C”-; “CC”-; “CXC”- and“CX3C”-chemokines). “CXC”-chemokines preferentially act on neutrophils.In contrast, “CC”-chemokines attract preferentially monocytes to sitesof inflammation. Monocyte infiltration is considered to be a key eventin a number of disease conditions (Gerard, C. and Rollins, B. J. (2001)Nat. Immunol 2, 108-115; Bhatia, M., et al., (2005) Pancreatology. 5,132-144; Kitamoto, S., Egashira, K., and Takeshita, A. (2003) JPharmacol Sci. 91, 192-196).

The MCP family, as one family of chemokines, consists of four members(MCP-1 to 4), displaying a preference for attracting monocytes butshowing differences in their potential (Luini, W., et al., (1994)Cytokine 6, 28-31; Uguccioni, M., et al., (1995) Eur J Immunol 25,64-68).

MCP-1 is a member of the β (C-C) subfamily of chemokines. In thisfamily, the 2 cysteins nearest to the amino terminus are adjacent toeach other (thus C-C proteins). As with many other C-C chemokines, theMCP-1 gene is located on chromosome 17 in humans. The cell surfacereceptors that bind MCP-1 are CCR2 and CCR5.

In the following both cDNA as well as amino acid sequences of MCP-1 areindicated:

Human MCP-1 (CCL2) (GeneBank Accession: M24545)

cDNA (300 bp) SEQ ID NO: 2   1 atgaaagtct ctgccgccct tctgtgcctgctgctcatag cagccacctt cattccccaa  61 gggctcgctc agccagatgc aatcaatgccccagtcacct gctgttataa cttcaccaat 121 aggaagatct cagtgcagag gctcgcgagctatagaagaa tcaccagcag caagtgtccc 181 aaagaagctg tgatcttcaa gaccattgtggccaaggaga tctgtgctga ccccaagcag 241 aagtgggttc aggattccat ggaccacctggacaagcaaa cccaaactcc gaagacttgaProtein (Signal Sequence in bold: 23 aa; Mature MCP-1: 76 aa)

SEQ ID NO: 1 KVSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT

Consistent with it being a member of the chemokine β family, MCP-1 hasbeen shown to chemoattract and activate monocytes in vitro atsubnanomolar concentrations. Elevated MCP-1 expression has been detectedin a variety of pathologic conditions that involve monocyte accumulationand activation, including a number of inflammatory and non-inflammatorydisease states, like rheumatoid arthritis, atherosclerosis, asthma anddelayed hypersensitivity reactions.

A number of studies have underlined in particular the crucial role ofMCP-1 for the development of atherosclerosis (Gu, L., et al., (1998)Mol. Cell 2, 275-281; Gosling, J., et al., (1999) J Clin. Invest 103,773-778); rheumatoid arthritis (Gong, J. H., et al., (1997) J Exp. Med186, 131-137; Ogata, H., et al., (1997) J Pathol. 182, 106-114);pancreatitis (Bhatia, M., et al., (2005) Am. J Physiol Gastrointest.Liver Physiol 288, G1259-G1265); Alzheimer's disease (Yamamoto, M., etal., (2005) Am. J Pathol. 166, 1475-1485); lung fibrosis (Inoshima, I.,et al., (2004) Am. J Physiol Lung Cell Mol. Physiol 286, L1038-L1044);renal fibrosis (Wada, T., et al., (2004) J Am. Soc. Nephrol. 15,940-948), and graft rejection (Saiura, A., et al., (2004) Arterioscler.Thromb. Vasc. Biol. 24, 1886-1890). Furthermore, MCP-1 might also play arole in gestosis (Katabuchi, H., et al., (2003) Med Electron Microsc.36, 253-262), as a paracrine factor in tumor development (Ohta, M., etal., (2003) Int. J Oncol. 22, 773-778; Li, S., et al., (2005) J Exp. Med202, 617-624), neuropathic pain (White, F. A., et al., (2005) Proc.Natl. Acad. Sci. U.S.A) and AIDS (Park, I. W., Wang, J. F., andGroopman, J. E. (2001) Blood 97, 352-358; Coll, B., et al., (2006)Cytokine 34, 51-55).

The mature form of human and rodent MCP-1 is post-translationallymodified by Glutaminyl Cyclase (QC) to possess an N-terminalpyroglutamyl (pGlu) residue.

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the intramolecularcyclization of N-terminal glutaminyl residues into pyroglutamic acid(5-oxo-proline, pGlu*) under liberation of ammonia and theintramolecular cyclization of N-terminal glutamyl residues intopyroglutamic acid under liberation of water.

The N-terminal pGlu modification makes the protein resistant againstN-terminal degradation by aminopeptidases, which is of importance, sincechemotactic potency of MCP-1 is mediated by its N-terminus (Van Damme,J., et al., (1999) Chem Immunol 72, 42-56). Artificial elongation ordegradation leads to a loss of function although MCP-1 still binds toits receptor (CCR2) (Proost, P., et al., (1998), J Immunol 160,4034-4041; Zhang, Y. J., et al., 1994, J Biol. Chem 269, 15918-15924;Masure, S., et al., 1995, J Interferon Cytokine Res. 15, 955-963;Hemmerich, S., et al., (1999) Biochemistry 38, 13013-13025).

Due to the major role of MCP-1 in a number of disease conditions, apotent diagnostic tool and an anti-MCP-1 strategy is required.

As mentioned above, compelling evidence points to a role of MCP 1 inAlzheimer's disease (AD) (Xia, M. Q. and Hyman, B. T. (1999) JNeurovirol. 5, 32-41). The presence of MCP-1 in senile plaques and inreactive microglia, the residential macrophages of the CNS, has beenobserved in brains of patients suffering from AD (Ishizuka, K., et al.,(1997) Psychiatry Clin. Neurosci. 51, 135-138. Stimulation of monocytesand microglia with Amyloid-β protein (Aβ) induces chemokine secretion invitro (Meda, L., et al., (1996) J Immunol 157, 1213-1218; Szczepanik, A.M., et al., (2001) J Neuroimmunol. 113, 49-62) andintracerebroventricular infusion of Aβ (1-42) into murine hippocampussignificantly increases MCP-1 in vivo. Moreover, Aβ deposits attract andactivate microglial cells and force them to produce inflammatorymediators such as MCP-1, which in turn leads to a feed back to inducefurther chemotaxis, activation and tissue damage. At the site of Aβdeposition, activated microglia also phagocytose Aβ peptides leading toan amplified activation (Rogers, J. and Lue, L. F. (2001) Neurochem.Int. 39, 333-340).

Examination of chemokine expression in the 3×Tg mouse model for ADrevealed that neuronal inflammation precedes plaque formation and MCP-1is upregulated by a factor of 11. Furthermore, the upregulation of MCP-1seems to correlate with the occurrence of first intracellular Aβdeposits (Janelsins, M. C., et al., (2005) J Neuroinflammation. 2, 23).Cross-breeding of the Tg2575 mouse model for AD with a MCP-1overexpressing mouse model has shown an increased microglia accumulationaround Aβ deposits and that this accumulation was accompanied byincreased amount of diffuse plaques compared to single-transgenic Tg2576littermates (Yamamoto, M., et al. (2005) Am. J Pathol. 166, 1475-1485).

MCP-1 levels are increased in CSF of AD patients and patients showingmild cognitive impairment (MCI) (Galimberti, D., et al., (2006) Arch.Neurol. 63, 538-543). Furthermore, MCP-1 shows an increased level inserum of patients with MCI and early AD (Clerici, F., et al., (2006)Neurobiol. Aging 27, 1763-1768).

Atherosclerotic lesions, which limit or obstruct coronary blood flow,are the major cause of ischemic heart disease related mortality,resulting in 500,000-600,000 deaths annually. Percutaneous transluminalcoronary angioplasty (PTCA) to open the obstructed artery was performedin over 550,000 patients in the U.S. and 945, 000+ patients worldwide in1996 (Lemaitre et al., 1996). A major limitation of this technique isthe problem of post-PTCA closure of the vessel, both immediately afterPTCA (acute occlusion) and in the long term (restenosis): 30% ofpatients with subtotal lesions and 50% of patients with chronic totallesions will go on to restenosis after angioplasty. Additionally,restenosis is a significant problem in patients undergoing saphenousvein bypass graft. The mechanism of acute occlusion appears to involveseveral factors and may result from vascular recoil with resultantclosure of the artery and/or deposition of blood platelets along thedamaged length of the newly opened blood vessel followed by formation ofa fibrin/red blood cell thrombus.

Restenosis after angioplasty is a more gradual process and involvesinitial formation of a subcritical thrombosis with release from adherentplatelets of cell derived growth factors with subsequent proliferationof intimal smooth muscle cells and local infiltration of inflammatorycells contributing to vascular hyperplasia. It is important to note thatmultiple processes, among those thrombosis, cell proliferation, cellmigration and inflammation each seem to contribute to the restenoticprocess.

In the U.S., a 30-50% restenosis rate translates to 120,000-200,000 U.S.patients at risk from restenosis. If only 80% of such patients electrepeated angioplasty (with the remaining 20% electing coronary arterybypass graft) and this is added to the cost of coronary artery bypassgraft for the remaining 20%, the total cost for restenosis easilyreaches into billions of dollars. Thus, successful prevention ofrestenosis could result not only in significant therapeutic benefit butalso in significant health care savings.

Although it is not clear whether elevated MCP-1 expression is the causeor consequence of the above diseases, therapeutic benefit resulted fromthe application of neutralizing antibodies in a number of animal models.

So far, prior art monoclonal antibodies were screened for their abilityto act as receptor antagonists. None of those target the immediatepyroglutamate carrying amino terminus of MCP-1 (=MCP1 N1pE). In thiscontext, it is important to note that deletion of amino acids 1-8 fromthe N-terminal region completely destroyed MCP-1 activity, suggestingthat the amino-terminal region is essential for activity.

It consequently follows, that antibodies directed against the MCP-1 N1pEcan play a role not only when investigating the expression and functionof MCP-1 but also in therapeutic and diagnostic applications inconnection with diseases or disturbances in which MCP-1 might beinvolved.

In view of the above, one object underlying the present invention is toprovide antibodies which are selectively binding to MCP-1 N1pE.

SUMMARY OF THE INVENTION

The present invention provides antibodies selectively binding to MCP-1N1pE, i.e. pyroglutamate MCP-1.

Preferably, monoclonal antibodies are provided.

The present invention pertains in particular to antibodies or variantsthereof, which are characterized in that they bind to the MCP-1 N1pEpeptide with a high affinity. Said high affinity means in the context ofthe present invention an affinity of a K_(D) value of 10⁻⁶ M or better,preferably a K_(D) value of 10⁻⁷ M or better, and even more preferably aK_(D) value of 10⁻⁸ M-10⁻¹² M.

Monoclonal antibodies of this type are preferably produced by hybridomacells. Hybridoma cells of this type were deposited on 06. May 2008 inthe Deutsche Sammlung von Mikroorganismen and Zellkulturen (GermanCollection of Microorganisms and Cell Cultures) GmbH, DSMZ,Inhoffenstrasse 7B, 38124 Braunschweig, Germany, in accordance with theBudapest Treaty, namely

DSM ACC 2905 (Hybridoma cell clone 348/1D4) 2906 (Hybridoma cell clone348/2C9) 2907 (Hybridoma cell clone 332/4B8) and 2908 (Hybridoma cellclone 332/4F8).

In particular, monoclonal antibodies are preferred, wherein selectivebinding means a binding to the pyroglutamate carrying amino terminus ofMCP-1.

Even preferred are monoclonal antibodies, wherein selective bindingmeans that the antibodies do not show any cross-reactivity with epitopesoutside the pyroglutamate carrying amino terminus of MCP-1 N1pE.

The amino terminus of MCP-1 N1pE is defined here as the first 1 to 10amino acids of the amino terminus of MCP-1 N1pE, preferably the first 1to 8 amino acids of the amino terminus of MCP N1pE, most preferably thefirst 1 to 4 amino acids of the amino terminus of MCP N1pE.

The invention relates further to the above hybridoma cell clones per sewhich possess the ability to produce and release such antibodies.

By means of the antibodies 332-4B8 (DSM ACC 2907), 332-4F8 (DSM ACC2908), 348-2C9(DSM ACC 2906) and 348-1D4 (DSM ACC 2905), the inventorsof the present application have, for the first time, made availablemonoclonal antibodies, as well as hybridoma cells which produce andrelease these antibodies, which make it possible to selectivelyrecognize and bind to, and consequently influence MCP-1 N1pE. Theantibodies consequently provide the physician and research worker with aversatile means, which is so far unique, for, on the one hand, detectingMCP-1 N1pE, both in cell culture and in the sample obtained from apatient, and, on the other hand, for potential manipulation of MCP-1N1pE, where appropriate, either using the antibody itself or usingspecific reagents which are coupled to it.

In this connection, the inventors of the present application haveascertained that the above antibodies 332-4B8, 332-4F8, 348-2C9 and348-1D4 bind selectively to polypeptides of MCP-1 N1pE starting withamino acids pE-P-D i.e. pyroglutamate-proline-aspartic acid.

The inventors have been able to demonstrate that MCP-1 N1pE is alsodetected in blood samples (serum, plasma) of mammals, especially ofmice, rats and humans, and that MCP-1 N1pE levels are elevated afterinflammatory stimuli, which can be reversed by application of selectiveglutminyl cyclase (QC) inhibitors (e.g.1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea-hydrochloride(see particularly the glutaminyl peptide cyclase inhibitors as disclosedin WO 2008/104580).

Consequently, these antibodies are outstandingly suitable for diagnosticand therapeutic purposes, with it being possible to achieve a widevariety of investigations and therapeutic effects.

Accordingly, a further embodiment of the invention relates to apharmaceutical composition comprising one of the above novel antibodies.Preferably said novel antibody is coupled to a cellular directedtherapeutic agent or diagnostic agent.

An antibody according to the invention, which is coupled to a means fordetection and thus indirectly to the relevant cells, thereby makes itpossible to detect these cells directly, for example using X-raydiagnostic/scintigraphic methods. In a corresponding manner, coupling toa therapeutically active agent can also make it possible to exert adirect and selective effect on MCP-1 N1pE carrying cells.

Further advantages will be evident from the description given below.

It will be understood that the features which are mentioned above, andthose which are still to be explained below, can be used not only in thecombinations which are in each case specified but also in othercombinations, or on their own, without departing from the scope of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1A: Binding characteristics of monoclonal antibody 332-4B8 to humanMCP-1 N1pE-38 determined with SPR analysis (Biacore 3000). Measurementwas performed by using HBS-EP as running buffer. Association took placefor 180 sec, followed by a 180 sec dissociation phase and 5 secregeneration with 0.1M HCl.

FIG. 1B: Binding characteristics of monoclonal antibody 332-4F8 to humanMCP-1 N1pE-38 determined with SPR analysis (Biacore 3000). Measurementwas performed by using HBS-EP as running buffer. Association took placefor 180 sec, followed by a 180 sec dissociation phase and 5 secregeneration with 0.1M HCl.

FIG. 1C: Binding characteristics of monoclonal antibody 348-2C9 to humanMCP-1 N1pE-38 determined with SPR analysis (Biacore 3000). Measurementwas performed by using HBS-EP as running buffer. Association took placefor 180 sec, followed by a 180 sec dissociation phase and 5 secregeneration with 0.1M HCl.

FIG. 1D: Binding characteristics of monoclonal antibody 348-1D4 to humanMCP-1 N1pE-38 determined with SPR analysis (Biacore 3000). Measurementwas performed by using HBS-EP as running buffer. Association took placefor 180 sec, followed by a 180 sec dissociation phase and 5 secregeneration with 0.1M HCl.

FIG. 2A: DotBlot analysis of monoclonal antibody 322-4B8 to human MCP-1N1pE-38 and MCP-1 3-38.

FIG. 2B: DotBlot analysis of monoclonal antibody 322-4F8 to human MCP-1N1pE-38 and MCP-1 3-38.

FIG. 2C: DotBlot analysis of monoclonal antibody 348-1D4 to human MCP-1N1pE-38 and MCP-1 3-38.

FIG. 2D: DotBlot analysis of monoclonal antibody 348-2C9 to human MCP-1N1pE-38 and MCP-1 3-38.

FIG. 3: PepSpot analysis of monoclonal antibodies 322-4B8, 322-4F8,348-1D4 and 348-2C9.

FIG. 4: Quantitative detection of recombinant human MCP-1 N1pE in anELISA by using the monoclonal anti MCP-1 antibodies 332-4B8, 348-2C9 and348-1D4.

FIG. 5: Detection of human MCP-1 N1pE from human serum by monoclonalantibodies 322-4B8, 348-1D4 and 348-2C9 in ELISA.

FIG. 6: Time dependent expression of hMCP-1 N1pE in NHDF cells,stimulated by OSM and IL1β.

FIG. 7: Concentration dependent reduction of hMCP-1 N1pE in the cellculture supernatant of LPS induced THP1 cells after application of QCI.

FIG. 8: Quantitative detection of recombinant mouse MCP-1 N1pE in anELISA by using the monoclonal anti MCP-1 antibodies 332-4B8, 348-2C9 and348-1D4

FIG. 9: Quantitative detection of native mouse MCP-1 N1pE in the cellculture supernatant of untreated and LPS induced RAW 264.7 cells.

FIG. 10: Concentration dependent reduction of mMCP-1 N1pE in the cellculture supernatant of LPS induced RAW 264.7 cells after application ofQCI.

FIG. 11: Quantitative detection of mMCP-1 N1pE in mouse serum afterdifferent time points of LPS treatment.

FIG. 12: Dilution Linearity of the quantitative detection of mMCP-1 N1pEin peritoneal lavage fluid from thioglycollate treated mice by ELISA.

FIG. 13: Comparison of Western Blot signals obtained for murine MCP1N1pE (A) and total murine MCP1 (B) with the corresponding ELISA Data formurine MCP1 N1pE (C).

FIG. 14: Quantitative detection of rat MCP-1 N1pE by the anti MCP-1 N1pEantibody 348-2C9 in an ELISA.

FIG. 15: Staining of MCP-1 N1pE in brain sections of rats with theantibodies 332-4B8, 348-1D4 and 348-2C9 after microinjection ofAβ(3-49), LPS or NaCl.

FIG. 16: Fitting curves of the binding heat evolved by titration of theantigen hMCP-1 N1pE-38 to the monoclonal antibodies A—348-1D4 andB—332-4B8.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS Definitions

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

The term “antibody” is used in the broadest sense and specificallycovers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity. The antibody may be an IgM, IgG(e.g. IgG1, IgG2, IgG3 or IgG4), IgD, IgA or IgE, for example.Preferably however, the antibody is not an IgM antibody.

“Antibody fragments” comprise a portion of an intact antibody, generallythe antigen binding or variable region of the intact antibody. Examplesof antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments:diabodies; single-chain antibody molecules; and multispecific antibodiesformed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to “polyclonalantibody” preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies can frequentlybe advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKöhler et al., Nature, 256:495 (1975), or may be made by generally wellknown recombinant DNA methods. The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include chimericantibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain a minimal sequence derived from a non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementarity-determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences.

These modifications are made to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986), Reichmann et al, Nature. 332:323-329 (1988): andPresta, Curr. Op. Struct. Biel., 2:593-596 (1992). The humanizedantibody includes a Primatized™ antibody wherein the antigen-bindingregion of the antibody is derived from an antibody produced byimmunizing macaque monkeys with the antigen of interest.

“Single-chain Fv” or “sFv” antibody fragments comprise the variableheavy chain (VH) and variable light chain (VL) domains of an antibody,wherein these domains are present in a single polypeptide chain.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the VH and VL domains which enables the sFv to form the desiredstructure for antigen binding. For a review of sFv see Pluckthun in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VD) in the samepolypeptide chain (VH-VD). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully inHollinger et al., Proc. Natl. Acad. Sol. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the antibody willbe purified (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. The isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, the isolated antibody will be prepared by at leastone purification step.

As used herein, the expressions “cell”, “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and culture derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, this will be clearfrom the context.

The terms “polypeptide”, “peptide”, and “protein”, as used herein, areinterchangeable and are defined to mean a biomolecule composed of aminoacids linked by a peptide bond.

“Homology” between two sequences is determined by sequence identity. Iftwo sequences which are to be compared with each other differ in length,sequence identity preferably relates to the percentage of the nucleotideresidues of the shorter sequence which are identical with the nucleotideresidues of the longer sequence. Sequence identity can be determinedconventionally with the use of computer programs such as the Bestfitprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, 575 Science DriveMadison, Wis. 53711). Bestfit utilizes the local homology algorithm ofSmith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489,in order to find the segment having the highest sequence identitybetween two sequences. When using Bestfit or another sequence alignmentprogram to determine whether a particular sequence has, for example, 95%identity with a reference sequence of the present invention, theparameters are preferably adjusted so that the percentage of identity iscalculated over the entire length of the reference sequence and homologygaps of up to 5% of the total number of the nucleotides in the referencesequence are permitted. When using Bestfit, the so-called optionalparameters are preferably left at their preset (“default”) values. Thedeviations appearing in the comparison between a given sequence and theabove-described sequences of the invention may be caused for instance byaddition, deletion, substitution, insertion or recombination. Such asequence comparison can preferably also be carried out with the program“fasta20u66” (version 2.0u66, September 1998 by William R. Pearson andthe University of Virginia; see also W. R. Pearson (1990), Methods inEnzymology 183, 63-98, appended examples andhttp://workbench.sdsc.edu/). For this purpose, the “default” parametersettings may be used.

As used herein, a “conservative change” refers to alterations that aresubstantially conformationally or antigenically neutral, producingminimal changes in the tertiary structure of the mutant polypeptides, orproducing minimal changes in the antigenic determinants of the mutantpolypeptides, respectively, as compared to the native protein. Whenreferring to the antibodies and antibody fragments of the invention, aconservative change means an amino acid substitution that does notrender the antibody incapable of binding to the subject receptor. One ofordinary skill in the art will be able to predict which amino acidsubstitutions can be made while maintaining a high probability of beingconformationally and antigenically neutral. Such guidance is provided,for example in Berzofsky, (1985) Science 229:932-940 and Bowie et al.(1990) Science 247: 1306-1310. Factors to be considered that affect theprobability of maintaining conformational and antigenic neutralityinclude, but are not limited to: (a) substitution of hydrophobic aminoacids is less likely to affect antigenicity because hydrophobic residuesare more likely to be located in a protein's interior; (b) substitutionof physiochemically similar, amino acids is less likely to affectconformation because the substituted amino acid structurally mimics thenative amino acid; and (c) alteration of evolutionarily conservedsequences is likely to adversely affect conformation as suchconservation suggests that the amino acid sequences may have functionalimportance. One of ordinary skill in the art will be able to assessalterations in protein conformation using well-known assays, such as,but not limited to microcomplement fixation methods (see, e.g. Wassermanet al. (1961) J. Immunol. 87:290-295; Levine et al. (1967) Meth.Enzymol. 11:928-936) and through binding studies usingconformation-dependent monoclonal antibodies (see, e.g. Lewis et al.(1983) Biochem. 22:948-954).

The terms “a”, “an” and “the” as used herein are defined to mean “one ormore” and include the plural unless the context is inappropriate.

The invention is explained in more detail below with the aid ofapplication examples and implementation examples as well as the Figures.

For diagnostic applications, the antibody typically will be labelledwith a detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

(a) Radioisotopes, such as 35S, 14C, 125I, 3H, and 131I. The antibodycan be labelled with the radioisotope using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Gütigen et al., Ed.,Wiley-Interscience. New York, N.Y. Pubs., (1991) for example andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody using thetechniques disclosed in Current Protocols in Immunology, supra forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available. Such enzyme generallycatalyses a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g, fireflyluciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase.O-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O′Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym (ed Langone & H. Van Vunakis),Academic Press, New York, 73: 147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g. orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.p-nitrophenyl-β-D-galactosidase) or the fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art.

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g. digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g. anti-digoxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

The inventive antibodies need not be labeled, and the presence thereofcan be detected using a labeled antibody, which binds to the inventiveantibodies.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays (Zola, MonoclonalAntibodies A Manual of Techniques, pp. 147-158 (CRC Press. Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of MCP-1 peptide in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. The second antibody may itself be labeled with adetectable moiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assay). For example, one preferable type of sandwichassay is an ELISA assay, in which case the detectable moiety is anenzyme.

For immunohistochemistry analysis, the tissue sample may be fresh orfrozen or may be embedded in paraffin and fixed with a preservative suchas formalin, for example.

Diagnostic Kits

As a matter of convenience, the antibody of the present invention can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labelled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g. asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g. a blocking buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied widelyto provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

The diagnostic kit according to the invention may contain a furtherbiologically active substance as described below. Especially preferredfor the use in the diagnostic kit are inhibitors of glutaminyl cyclase.

The diagnostic kit of the invention is especially useful for thedetection and diagnosis of MCP-1-related diseases and conditionsselected from the group consisting of inflammatory diseases selectedfrom

-   -   a. neurodegenerative diseases, e.g. mild cognitive impairment        (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome,        Familial British Dementia, Familial Danish Dementia, multiple        sclerosis,    -   b. chronic and acute inflammations, e.g. rheumatoid arthritis,        atherosclerosis, restenosis, pancreatitis,    -   c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis,    -   d. cancer, e.g. cancer/hemangioendothelioma proliferation,        gastric carcinomas,    -   e. metabolic diseases, e.g. hypertension,    -   f. and other inflammatory diseases, e.g. neuropathic pain, graft        rejection/graft failure/graft vasculopathy, HIV infections/AIDS,        gestosis, tuberous sclerosis.

Preferably, the antibody according to the present invention isespecially useful in a diagnostic method to detect MCP-1-relateddisease, e.g. atheroschlerosis, rheumatoid arthritis, asthma, delayedhypersensitivity reactions, pancreatitis, Alzheimer's disease, lungfibrosis, renal fibrosis, gestosis, graft rejection, neuropathic pain,AIDS and tumors.

Most preferably, the diagnostic kit of the invention is useful for thedetection and diagnosis of Alzheimer's disease, or also most preferablya disease selected from atherosclerosis, rheumatoid arthritis,restenosis and pancreatitis, in particular Alzheimer's disease orrheumatoid arthritis.

Preferred according to the present invention is a monoclonal antibody.

More preferably according to the present invention is a monoclonalantibody, wherein the variable part of the light chain of said antibodyhas a nucleotide sequence selected from SEQ ID NOs: 33, 37 and 41, or anamino acid sequence selected from SEQ ID NOs: 34, 38 and 42.

Alternatively preferred according to the present invention is amonoclonal antibody, wherein the variable part of the heavy chain ofsaid antibody has a nucleotide sequence selected from SEQ ID NOs: 35, 39and 43, or an amino acid sequence selected from SEQ ID NOs: 36, 40 and44.

Further preferred according to the present invention is the monoclonalantibody, wherein the variable part of the light chain of said antibodyhas the nucleotide sequence of SEQ ID NO: 33 or the amino acid sequenceof SEQ ID NO: 34, and wherein the variable part of the heavy chain ofsaid antibody has the nucleotide sequence of SEQ ID NO: 35, or the aminoacid sequence of SEQ ID NO: 36.

Also preferred according to the present invention is the monoclonalantibody, wherein the variable part of the light chain of said antibodyhas the nucleotide sequence of SEQ ID NO: 37 or the amino acid sequenceof SEQ ID NO: 38, and wherein the variable part of the heavy chain ofsaid antibody has the nucleotide sequence of SEQ ID NO: 39, or the aminoacid sequence of SEQ ID NO: 40.

Even preferred according to the present invention is the monoclonalantibody, wherein the variable part of the light chain of said antibodyhas the nucleotide sequence of SEQ ID NO: 41 or the amino acid sequenceof SEQ ID NO: 42, and wherein the variable part of the heavy chain ofsaid antibody has the nucleotide sequence of SEQ ID NO: 43, or the aminoacid sequence of SEQ ID NO: 44.

In particular preferred is a monoclonal antibody, which is produced by ahybridoma cell line selected from the following group

348/1D4 (Deposit No. DSM ACC 2905) 348/2C9 (Deposit No. DSM ACC 2906)332/4B8 (Deposit No. DSM ACC 2907) 332/4F8 (Deposit No. DSM ACC 2908)

According to a further preferred embodiment, the antibody can behumanised or is a chimeric antibody or is a human antibody.

Further, the antibody as selected from the above-mentioned group canalso be a functional variant of said group.

In the context of the present invention a “functional variant” of theinventive antibody is an antibody which retains the binding capacities,in particular binding capacities with high affinity to a MCP-1 N1pE-38or functional variant thereof. The provision of such functional variantsis known in the art and encompasses the above-mentioned possibilities,which were indicated under the definition of antibodies and fragmentsthereof.

In a preferred embodiment, the antibody is an antibody fragment, asdefined above.

In a further preferred embodiment, the antibody of the invention is anantibody which has the complementarity-determining regions (CDRs) of theabove-defined antibodies. Preferably, the antibody can be labeled;possible labels are those as mentioned above and all those known to aperson skilled in the art of diagnostic uses of antibodies inparticular.

The present invention further relates to a light chain variable regioncomprising an nucleic acid sequence that is 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to asequence selected from SEQ ID NOs: 33, 37 or 41, or a functional partthereof.

The present invention further relates to a heavy chain variable regioncomprising an nucleic acid sequence that is 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to asequence selected from SEQ ID NOs: 35, 39 or 43, or a functional partthereof.

Further preferred according to the present invention is a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof, which antibody comprises a light chain variable domaincomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequenceselected from SEQ ID NOs: 34, 38 or 42.

Even preferred according to the present invention is a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof, which antibody comprises a heavy chain variable domaincomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequenceselected from SEQ ID NOs: 36, 40 or 44.

Moreover, the present invention relates to a monoclonal antibodyincluding any functionally equivalent antibody or functional partsthereof, wherein the variable part of the light chain of said antibodycomprises an amino acid sequence selected from SEQ ID NOs: 34, 38 and 42and/or wherein the variable part of the heavy chain of said antibodycomprises an amino acid sequence selected from SEQ ID NOs: 36, 40 and44, wherein the antibody has been altered by introducing at least one,at least two, or at least 3 or more conservative substitutions into atleast one of the sequences of SEQ ID NOs: 34, 36, 38, 40, 42 and 44,wherein the antibody essentially maintains its full functionality.

The present invention further relates to an isolated polynucleotideencoding the light chain variable region of the monoclonal antibodiespresented herein, wherein said isolated polynucleotide comprises anucleic acid sequence selected from SEQ ID Nos: 33, 37 and 41.

The present invention also relates to an isolated polynucleotideencoding the heavy chain variable region of the monoclonal antibodiespresented herein, wherein said isolated polynucleotide comprises anucleic acid sequence selected from SEQ ID Nos: 35, 39 and 43.

Moreover, the present invention relates to an isolated peptide of thelight chain variable region of the monoclonal antibodies presentedherein, wherein said isolated peptide comprises an amino acid sequenceselected from SEQ ID Nos: 34, 38 and 42.

Moreover, the present invention relates to an isolated peptide of theheavy chain variable region of the monoclonal antibodies presentedherein, wherein said isolated peptide comprises an amino acid sequenceselected from SEQ ID Nos: 36, 40 and 44.

In a further preferred embodiment, the present invention relates to anoligonucleotide selected from the group consisting of SEQ ID NOs: 7 to32.

Preferably, the antibody is immobilised on a solid phase.

The present invention also relates to a composition which comprises theantibody as defined above. In particular, said composition is acomposition for a diagnostic use, especially for the diagnosis ofMCP-1-related diseases, in particular by detection of MCP-1 N1pE orvariants thereof in a biological sample.

In another embodiment, the antibody according to the invention and asdescribed herein before or a fragment thereof, exhibits a bindingaffinity to MCP-1 N1pE, which is at least 2 times, particularly at least4 times, particularly at least 10 times, particularly at least 15 times,more particularly at least 20 times, but especially at least 25 timeshigher than the binding affinity of conventional antibodies.

In still another embodiment, a chimeric antibody or a fragment thereof,or a humanized antibody or a fragment thereof is provided as describedherein before, which antibody substantially binds to MCP-1 N1pE in themammalian, particularly the human brain but, preferably, does not showany significant cross-reactivity with MCP-1 N1pE, in particular withMCP-1 N1pE 3-38.

The present invention relates also to humanized forms of the antibodiesas defined above, compositions comprising said humanized antibodies andthe use of said compositions for the treatment of MCP-1-relateddiseases, especially for the treatment of Alzheimer's disease in amammal, in particular in a human.

The present invention is also directed to the following hybridoma celllines

DSM ACC 2905 (Hybridoma cell clone 348/1D4) DSM ACC 2906 (Hybridoma cellclone 348/2C9) DSM ACC 2907 (Hybridoma cell clone 332/4B8) and DSM ACC2908 (Hybridoma cell clone 332/4F8).

The present invention also pertains to the use of the antibody or thecomposition comprising the antibody, both as defined above, in an invitro diagnostic method. In particular, this diagnostic method isdirected to diagnosis of MCP-1-related diseases, especially by detectingan MCP-1 N1pE or variants thereof in a biological sample.

Preferably, said sample is a serum sample.

According to another preferred embodiment, said sample is a liquor,cerebrospinal fluid (CSF) or synovial fluid sample.

In a particularly preferred embodiment, the present invention pertainsto the following method:

In vitro or in situ diagnostic method for the diagnosis of anMCP-1-related disease or condition, comprising the following steps:

-   -   a) contacting an antibody according to the invention with a        sample, preferably selected from a serum, liquor or CSF sample,        most preferably a serum sample; or a specific body part or body        area of a subject suspected to be afflicted with said condition        or disease, and    -   b) detecting binding of the antibody to an MCP-1 N1pE peptide,        from the sample.

More particularly, the invention relates to a method of diagnosis of anMCP-1-related disease or condition, comprising detecting theimmunospecific binding of an antibody or an active fragment thereof toan MCP-1 N1pE peptide, in a sample or in situ which includes the stepsof

-   -   (a) bringing the sample or a specific body part or body area        suspected to contain the MCP-1 peptide into contact with an        antibody, particularly a monoclonal antibody according to the        present invention, or a chimeric antibody or a fragment thereof,        or a humanized antibody or a fragment thereof according to the        invention and as described herein before, and/or a functional        part thereof;    -   (b) allowing the antibody and/or a functional part thereof, to        bind to the MCP-1 N1pE peptide to form an immunological complex;    -   (c) detecting the formation of the immunological complex; and    -   (d) correlating the presence or absence of the immunological        complex with the presence or absence of MCP-1 N1pE peptide in        the sample or specific body part or area.

The aforementioned diagnostic methods are especially useful for thedetection and diagnosis of MCP-1-related diseases and conditionsselected from the group consisting of inflammatory diseases selectedfrom

-   -   a. neurodegenerative diseases, e.g. mild cognitive impairment        (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome,        Familial British Dementia, Familial Danish Dementia, multiple        sclerosis,    -   b. chronic and acute inflammations, e.g. rheumatoid arthritis,        atherosclerosis, restenosis, pancreatitis,    -   c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis,    -   d. cancer, e.g. cancer/hemangioendothelioma proliferation,        gastric carcinomas,    -   e. metabolic diseases, e.g. hypertension,    -   f. and other inflammatory diseases, e.g. neuropathic pain, graft        rejection/graft failure/graft vasculopathy, HIV infections/AIDS,        gestosis, tuberous sclerosis.

Preferably, the aforementioned diagnostic methods are useful to detectMCP-1-related disease, e.g. atheroschlerosis, rheumatoid arthritis,asthma, delayed hypersensitivity reactions, pancreatitis, Alzheimer'sdisease, lung fibrosis, renal fibrosis, gestosis, graft rejection,neuropathic pain, AIDS and tumors.

Most preferably, the aforementioned diagnostic methods are useful forthe detection and diagnosis of Alzheimer's disease, or also mostpreferably a disease selected from atherosclerosis, rheumatoidarthritis, restenosis and pancreatitis, in particular Alzheimer'sdisease or rheumatoid arthritis.

In still another embodiment, the invention relates to a compositioncomprising the antibody according to the invention, or a chimericantibody or a fragment thereof, or a humanized antibody or a fragmentthereof according to the invention and as described herein beforeincluding any functionally equivalent antibody or any derivative orfunctional parts thereof, in a therapeutically effective amount, inparticular a composition which is a pharmaceutical compositionoptionally further comprising a pharmaceutically acceptable carrier.

In another embodiment of the invention, said composition comprises theantibody in a therapeutically effective amount.

Further comprised by the invention is a mixture comprising an antibody,particularly a monoclonal antibody according to the invention, or achimeric antibody or a fragment thereof, or a humanized antibody or afragment thereof according to the invention and as described hereinbefore including any functionally equivalent antibody or any derivativeor functional parts thereof, in a therapeutically effective amount and,optionally, a further biologically active substance and/or apharmaceutically acceptable carrier and/or a diluent and/or anexcipient.

In particular, the invention relates to a mixture, wherein the furtherbiologically active substance is a compound used in the medication of agroup of diseases and disorders associated with MCP-1, such as aninflammatory diseases selected from

-   -   a. neurodegenerative diseases, e.g. mild cognitive impairment        (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome,        Familial British Dementia, Familial Danish Dementia, multiple        sclerosis,    -   b. chronic and acute inflammations, e.g. rheumatoid arthritis,        atherosclerosis, restenosis, pancreatitis,    -   c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis,    -   d. cancer, e.g. cancer/hemangioendothelioma proliferation,        gastric carcinomas,    -   e. metabolic diseases, e.g. hypertension, and    -   f. other inflammatory diseases, e.g. neuropathic pain, graft        rejection/graft failure/graft vasculopathy, HIV infections/AIDS,        gestosis, tuberous sclerosis.

The other biologically active substance or compound may exert itsbiological effect by the same or a similar mechanism as the antibodyaccording to the invention or by an unrelated mechanism of action or bya multiplicity of related and/or unrelated mechanisms of action.

Generally, the other biologically active compound may includeneutron-transmission enhancers, psychotherapeutic drugs, acetylcholineesterase inhibitors, calcium-channel blockers, biogenic amines,benzodiazepine tranquillizers, acetylcholine synthesis, storage orrelease enhancers, acetylcholine postsynaptic receptor agonists,monoamine oxidase-A or -B inhibitors, N-methyl-D-aspartate glutamatereceptor antagonists, non-steroidal anti-inflammatory drugs,antioxidants, serotonergic receptor antagonists, CCR2 receptorantagonists and MCP-1 antibodies. With MCP-1 antibodies as otherbiologically active agent are meant such antibodies, which are bindingnative MCP-1, i.e. where the N-terminal Glu residue is not cyclized topGlu.

More particularly, the invention relates to a mixture comprising atleast one compound selected from the group consisting of compoundseffective against oxidative stress, anti-apoptotic compounds, metalchelators, inhibitors of DNA repair such as pirenzepin and metabolites,3-amino-1-propanesulfonic acid (3 APS), 1,3-propanedisulfonate (1,3PDS),α-secretase activators, β- and γ-secretase inhibitors, tau proteins,neurotransmitter, β-sheet breakers, attractants for amyloid betaclearing/depleting cellular components, inhibitors of N-terminaltruncated amyloid beta peptides including pyroglutamated amyloid beta3-42, such as inhibitors of glutaminyl cyclase, anti-inflammatorymolecules, or cholinesterase inhibitors (ChEIs) such as tacrine,rivastigmine, donepezil, and/or galantamine, M1 agonists and other drugsincluding any amyloid or tau modifying drug and nutritive supplements,and nutritive supplements, together with an antibody according to thepresent invention and, optionally, a pharmaceutically acceptable carrierand/or a diluent and/or an excipient.

The invention further relates to a mixture, wherein the compound is acholinesterase inhibitor (ChEIs), particularly a mixture, wherein thecompound is one selected from the group consisting of tacrine,rivastigmine, donepezil, galantamine, niacin and memantine.

In a further embodiment, the mixtures according to the invention maycomprise niacin or memantine together with an antibody according to thepresent invention and, optionally, a pharmaceutically acceptable carrierand/or a diluent and/or an excipient.

In a further embodiment, the mixtures according to the invention maycomprise a glutaminyl cyclase inhibitor together with an antibodyaccording to the present invention and, optionally, a pharmaceuticallyacceptable carrier and/or a diluent and/or an excipient.

Preferred inhibitors of glutaminyl cyclase are described in WO2005/075436, in particular examples 1-141 as shown on pp. 31-40. Thesynthesis of examples 1-141 is shown on pp. 40-48 of WO 2005/075436. Thedisclosure of WO 2005/075436 regarding examples 1-141, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/055945, in particular examples 1-473 as shown on pp. 46-155. Thesynthesis of examples 1-473 is shown on pp. 156-192 of WO 2008/055945.The disclosure of WO 2008/055945 regarding examples 1-473, theirsynthesis and their use as glutaminyl cyclase inhibitors is incorporatedherein by reference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/055947, in particular examples 1-345 as shown on pp. 53-118. Thesynthesis of examples 1-345 is shown on pp. 119-133 of WO 2008/055947.The disclosure of WO 2008/055947 regarding examples 1-345, theirsynthesis and their use as glutaminyl cyclase inhibitors is incorporatedherein by reference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/055950, in particular examples 1-212 as shown on pp. 57-120. Thesynthesis of examples 1-212 is shown on pp. 121-128 of WO 2008/055950.The disclosure of WO 2008/055950 regarding examples 1-212, theirsynthesis and their use as glutaminyl cyclase inhibitors is incorporatedherein by reference.

Further preferred inhibitors of glutaminyl cyclase are described inWO2008/065141, in particular examples 1-25 as shown on pp. 56-59. Thesynthesis of examples 1-25 is shown on pp. 60-67 of WO2008/065141. Thedisclosure of WO2008/065141 regarding examples 1-25, their synthesis andtheir use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/110523, in particular examples 1-27 as shown on pp. 55-59. Thesynthesis of examples 1-27 is shown on pp. 59-71 of WO 2008/110523. Thedisclosure of WO 2008/110523 regarding examples 1-27, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/128981, in particular examples 1-18 as shown on pp. 62-65. Thesynthesis of examples 1-18 is shown on pp. 65-74 of WO 2008/128981. Thedisclosure of WO 2008/128981 regarding examples 1-18, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/128982, in particular examples 1-44 as shown on pp. 61-67. Thesynthesis of examples 1-44 is shown on pp. 68-83 of WO 2008/128982. Thedisclosure of WO 2008/128982 regarding examples 1-44, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/128983, in particular examples 1-30 as shown on pp. 64-68. Thesynthesis of examples 1-30 is shown on pp. 68-80 of WO 2008/128983. Thedisclosure of WO 2008/128983 regarding examples 1-30, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/128984, in particular examples 1-36 as shown on pp. 63-69. Thesynthesis of examples 1-36 is shown on pp. 69-81 of WO 2008/128984. Thedisclosure of WO 2008/128984 regarding examples 1-36, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/128985, in particular examples 1-71 as shown on pp. 66-76. Thesynthesis of examples 1-71 is shown on pp. 76-98 of WO 2008/128985. Thedisclosure of WO 2008/128985 regarding examples 1-71, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/128986, in particular examples 1-7 as shown on pp. 65-66. Thesynthesis of examples 1-7 is shown on pp. 66-73 of WO 2008/128986. Thedisclosure of WO 2008/128986 regarding examples 1-7, their synthesis andtheir use as glutaminyl cyclase inhibitors is incorporated herein byreference.

In still another embodiment of the invention mixtures are provided thatcomprise “atypical antipsychotics” such as, for example clozapine,ziprasidone, risperidone, aripiprazole or olanzapine for the treatmentof positive and negative psychotic symptoms including hallucinations,delusions, thought disorders (manifested by marked incoherence,derailment, tangentiality), and bizarre or disorganized behavior, aswell as anhedonia, flattened affect, apathy, and social withdrawal,together with an antibody, particularly a monoclonal antibody accordingto the invention, but particularly a chimeric antibody or a fragmentthereof, or a humanized antibody or a fragment thereof according to theinvention and as described herein and, optionally, a pharmaceuticallyacceptable carrier and/or a diluent and/or an excipient.

In a specific embodiment of the invention, the compositions and mixturesaccording to the invention and as described herein before comprise theantibody and the biologically active substance, respectively, in atherapeutically effective amount.

Other compounds that can be suitably used in mixtures in combinationwith the antibody according to the present invention are described in WO2008/065141 (see especially pages 37/38), including PEP-inhibitors (pp.43/44), LiCl, inhibitors of dipeptidyl aminopeptidases, preferablyinhibitors of DP IV or DP IV-like enzymes (see pp. 48/49);acetylcholinesterase (ACE) inhibitors (see p. 47), PIMT enhancers,inhibitors of beta secretases (see p. 41), inhibitors of gammasecretases (see pp. 41/42), inhibitors of neutral endopeptidase,inhibitors of phosphodiesterase-4 (PDE-4) (see pp. 42/43), TNFalphainhibitors, muscarinic M1 receptor antagonists (see p. 46), NMDAreceptor antagonists (see pp. 47/48), sigma-1 receptor inhibitors,histamine H3 antagonists (se p. 43), immunomodulatory agents,immunosuppressive agents or an agent selected from the group consistingof antegren (natalizumab), Neurelan (fampridine-SR), campath(alemtuzumab), IR 208, NBI 5788/MSP 771 (tiplimotide), paclitaxel,Anergix.MS (AG 284), SH636, Differin (CD 271, adapalene), BAY 361677(interleukin-4), matrix-metalloproteinase-inhibitors (e.g. BB 76163),interferon-tau (trophoblastin) and SAIK-MS; beta-amyloid antibodies (seep. 44), cysteine protease inhibitors (see p. 44); MCP-1 antagonists (seepp. 44/45), amyloid protein deposition inhibitors (see p. 42) and betaamyloid synthesis inhibitors (see p. 42), which document is incorporatedherein by reference.

In another embodiment, the invention relates to a mixture comprising theantibody, particularly a monoclonal antibody according to the invention,or a chimeric antibody or a fragment thereof, or a humanized antibody ora fragment thereof according to the invention and as described hereinbefore and/or the biologically active substance in a therapeuticallyeffective amount.

The invention further relates to the use of an antibody, particularly amonoclonal antibody according to the invention, but particularly achimeric antibody or a fragment thereof, or a humanized antibody or afragment thereof according to the invention and as described hereinbefore and/or a functional part thereof and/or a pharmaceuticalcomposition, or a mixture comprising said antibody, for the preparationof a medicament for treating or alleviating the effects of a group ofdiseases and disorders associated with MCP-1 such as diseases including,but not limited to, neurological disorders such as Alzheimer's Disease(AD), Lewy body dementia, Down's syndrome, hereditary cerebralhemorrhage with amyloidosis (Dutch type); the Guam Parkinson-Dementiacomplex; as well as other diseases which are based on or associated withamyloid-like proteins such as progressive supranuclear palsy, multiplesclerosis; Creutzfeld Jacob disease, Parkinson's disease, HIV-relateddementia, ALS (amyotropic lateral sclerosis), Adult Onset Diabetes;senile cardiac amyloidosis; endocrine tumors, and others, includingmacular degeneration.

More preferably, the present invention relates to the use of anantibody, particularly a monoclonal antibody according to the invention,but particularly a chimeric antibody or a fragment thereof, or ahumanized antibody or a fragment thereof according to the invention andas described herein before and/or a functional part thereof and/or apharmaceutical composition, or a mixture comprising said antibody, forthe preparation of a medicament for treating or alleviating the effectsof an inflammatory diseases selected from

-   -   a. neurodegenerative diseases, e.g. mild cognitive impairment        (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome,        Familial British Dementia, Familial Danish Dementia, multiple        sclerosis,    -   b. chronic and acute inflammations, e.g. rheumatoid arthritis,        atherosclerosis, restenosis, pancreatitis,    -   c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis,    -   d. cancer, e.g. cancer/hemangioendothelioma proliferation,        gastric carcinomas,    -   e. metabolic diseases, e.g. hypertension, and    -   f. other inflammatory diseases, e.g. neuropathic pain, graft        rejection/graft failure/graft vasculopathy, HIV infections/AIDS,        gestosis, tuberous sclerosis.

Also comprised by the present invention is a method for the preparationof an antibody, particularly a monoclonal antibody according to theinvention, but particularly a chimeric antibody or a fragment thereof,or a humanized antibody or a fragment thereof according to the inventionand as described herein before and/or a functional part thereof and/or apharmaceutical composition, or a mixture comprising said antibody and/ora functional part thereof, particularly in a therapeutically effectiveamount, for use in a method of preventing, treating or alleviating theeffects of a group of diseases and disorders associated with MCP-1 asdefined above comprising formulating an antibody, particularly amonoclonal antibody according to the invention, but particularly achimeric antibody or a fragment thereof, or a humanized antibody or afragment thereof according to the invention in a pharmaceuticallyacceptable form.

Further comprised by the present invention is a method for preventing,treating or alleviating the effects of a group of diseases and disordersassociated with MCP-1 as defined above by administering an antibodyand/or a functional part thereof, but particularly a humanized antibodyand/or a functional part thereof, or a composition or mixture comprisingsuch an antibody and/or a functional part thereof, to an animal or ahuman affected by such a disorder comprising administering the antibodyin a therapeutically effective amount.

Administration and Dosage

The antibody is preferably administered to a mammal in a carrier;preferably a pharmaceutically-acceptable carrier. Suitable carriers andtheir formulations are described in Remington's Pharmaceutical Sciences,18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990;and Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000. Typically, an appropriate amount of a pharmaceuticallyacceptable salt is used in the formulation to render the formulationisotonic. Examples of the carrier include saline, Ringer's solution anddextrose solution. The pH of the solution is preferably from about 5 toabout 8, and more preferably from about 7 to about 7.5. Further carriersinclude sustained release preparations such as semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibody can be administered to the mammal by injection (e.g.,systemic, intravenous, intraperitoneal, subcutaneous, intramuscular,intraportal, intracerebral, intracerebral-ventricular, and intranasal),or by other methods, such as infusion, which ensure its delivery to thebloodstream in an effective form. The antibody may also be administeredby isolated perfusion techniques, such as isolated tissue perfusion, toexert local therapeutic effects. Intravenous injection is preferred.

Effective dosages and schedules for administering the antibody may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibody that must be administered will vary depending on, forexample, the mammal that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered to the mammal. Guidance in selecting appropriatedoses for antibody is found in the literature on therapeutic uses ofantibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al.,eds., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and pp.303-357; Smith et al. Antibodies in Human Diagnosis and Therapy, Haberet al., eds., Raven Press, New York, 1977, pp. 365-389. A typical dailydosage of the antibody used alone might range from about 1 μg/kg to upto 100 mg/kg of body weight or more per day, depending on the factorsmentioned above. Generally, any of the following doses may be used: adose of at least about 50 mg/kg body weight; at least about 10 mg/kgbody weight; at least about 3 mg/kg body weight; at least about 1 mg/kgbody weight; at least about 750 μg/kg body weight; at least about 500μg/kg body weight; at least about 250 ug/kg body weight; at least about100 μg/kg body weight; at least about 50 μg/kg body weight; at leastabout 10 μg/kg body weight; at least about 1 μg/kg body weight, or more,is administered. Antibodies may be administered at lower doses or lessfrequent at the beginning of the treatment to avoid potential sideeffect.

In some embodiments, more than one antibody may be present. Suchcompositions may contain at least one, at least two, at least three, atleast four, at least five different antibodies (including polypeptides)of the invention.

The antibody may also be administered to the mammal in combination witheffective amounts of one or more other therapeutic agents. The antibodymay be administered sequentially or concurrently with the one or moreother therapeutic agents. The amounts of antibody and therapeutic agentdepend, for example, on what type of drugs are used, the pathologicalcondition being treated, and the scheduling and routes of administrationbut would generally be less than if each were used individually.

Following administration of antibody to the mammal, the mammal'sphysiological condition can be monitored in various ways well known tothe skilled practitioner. The above principles of administration anddosage can be adapted for polypeptides described herein.

A polynucleotide encoding an antibody or a polypeptide described hereinmay also be used for delivery and expression of the antibody or thepolypeptide in a desired cell. It is apparent that an expression vectorcan be used to direct expression of the antibody. The expression vectorcan be administered systemically, intraperitoneally, intravenously,intramuscularly, subcutaneously, intrathecally, intraventricularly,orally, enterally, parenterally, intranasally, dermally, or byinhalation. For example, administration of expression vectors includeslocal or systemic administration, including injection, oraladministration, particle gun or catheterized administration, and topicaladministration. One skilled in the art is familiar with administrationof expression vectors to obtain expression of an exogenous protein invivo. See, e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.

Targeted delivery of therapeutic compositions comprising apolynucleotide encoding an antibody of the invention can also be used.Receptor-mediated DNA delivery techniques are described in, for example,Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., GeneTherapeutics: Methods And Applications Of Direct Gene Transfer (J. A.Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al.,J. Biol Chem. (1994) 269:542; Zenke et al, Proc. Natl. Acad. Sd. (USA)(1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg toabout 100 μg of DNA can also be used during a gene therapy protocol. Thetherapeutic polynucleotides and polypeptides of the present inventioncan be delivered using gene delivery vehicles. The gene delivery vehiclecan be of viral or non-viral origin (see generally, Jolly, Cancer GeneTherapy (1994) 1:51; Kirnura, Human Gene Therapy (1994) 5:845; Connelly,Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994)6:148). Expression of such coding sequences can be induced usingendogenous mammalian or heterologous promoters. Expression of the codingsequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740; 4,777,127; GB Patent No. 2,200,651; and EP 0 345242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semlikiforest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373;ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923;ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus(AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769;WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administrationof DNA linked to killed adenovirus as described in Curiel, Hum. GeneTher. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat.No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP 0 524 968. Additionalapproaches are described in Philip, MoI Cell Biol (1994) 14:2411, and inWoffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

In some embodiments, the numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

All publications, patents, patent applications, and other referencescited in this application are incorporated herein by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application or other reference wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes. Citation of a reference herein shallnot be construed as an admission that such is prior art to the presentinvention.

Having described the invention in detail, it will be apparent thatmodifications, variations, and equivalent embodiments are possiblewithout departing the scope of the invention defined in the appendedclaims. Furthermore, it should be appreciated that all examples in thepresent disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present invention. It should be appreciated by those of skill in theart that the techniques disclosed in the examples that follow representapproaches the inventors have found function well in the practice of theinvention, and thus can be considered to constitute examples of modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 Preparing and Characterizing Monoclonal Antibodies which areDirected Against MCP-1 N1pE

The aim was the generation of monoclonal antibodies reactive with thepE-P-D-A (SEQ ID No:3) containing amino acid sequence at the aminoterminus of the peptide MCP-1 N1pE-38 (SEQ ID No:4) (which is MCP-1 N1pEwith the first 38 amino acids starting from the N-terminus), but notreactive with the peptide MCP-1 D3-38 (SEQ ID No:5) which is the samemolecule as MCP-1 N1pE-38 but lacking pE and P at the amino terminus.

For immunisations the peptide pE-P-D-A-I-N-A-P-V-C amide (human MCP-1N1pE-9 (SEQ ID No:6)) was used. This low molecular weight antigen wasconjugated to Bovine Serum Albumin (Purified Fraction V BSA; Pierce) ascarrier protein using Sulfo-MBS (Pierce) as cross-linker.

To generate the monoclonal antibodies 8-week-old female BALB/c mice wereimmunised with the peptide-BSA-conjugate in two different immunisationprocedures as shown in table 1:

TABLE 1 Immunisation protocol for generation of monoclonal MCP-1 N1pEantibodies Long Time Short Time Immunisation Immunisation Doses (Day)(Day) Injection (μg/mouse) Adjuvant 1 1 Priming (i.p.) 100 TiterMax GoldAdjuvant (Sigma Alrich, St. Louis, USA) 30 14 Boost (i.p.) 100 TiterMaxGold Adjuvant (Sigma) 60 21 Boost (i.p.) 50 Incomplete Freund's Adjuvant(Sigma) 90 28 Boost (i.p.) 50 Incomplete Freund's Adjuvant (Sigma) 12635 Boost (i.v.) 50 PBS 129 38 Fusion

For example, 100 μg peptide corresponds to 50 μl ofpeptide-BSA-conjugate dissolved in PBS. The peptide-BSA-conjugate wasemulsified in an equal volume of TiterMax Gold Adjuvant (Sigma) orincomplete Freund's adjuvant and injected as stable emulsionintraperitoneally (i.p.). Three days before the fusion experiment wasperformed, each mouse received a total dose of 50 μg peptide (25 μlpeptide-BSA-conjugate) dissolved in 25 μl PBS given as i.v. injection.

The presence of the desired antibody was detected in the sera of therecipient prior to the final booster dose using the enzyme linkedimmunosorbent assay (ELISA) with human MCP-1 N1pE-9 as immobilizedantigen. The specific antibody titres were greater than 1:200000.

For fusion procedures, 6×10⁷ spleen cells from the immunised mice and2×10⁷ cells from mouse myeloma cell line SP2/0 were incubated with 1.2ml of 50% polyethylene glycol (Sigma) for 30 seconds at 37° C. Afterwashing, the cells were seeded in four 96-well cell culture plates.Hybrid clones were selected by growing in HAT medium [RPMI 1640 culturemedium (Biochrom, Berlin, DE) supplemented with 20% fetal calf serum(PAN Biotech GmbH, Aigenbach DE) and HAT-Supplement (50×; PAN)].

The culture supernatants were primarily screened for antigen specificIgG antibodies two weeks after fusion. The presence of an antigenspecific antibody in the culture supernatants was measured by itsbinding to the following peptides:

-   -   human MCP-1 1-9,    -   human MCP-1 N1pE-38 or    -   human MCP-1 D3-38,        especially, attached directly to the wells of a 96-well plate.        The antibody binding was quantified by adding the relevant        anti-species immunoglobulin (rabbit anti-mouse IgG (HRP)        Fc-specific antibody, Pierce, Rockford, USA) to which an enzyme        is bound, followed by a chromogenic substrate to that enzyme.        Fresh culture medium and a dilution of the polyclonal mouse        antiserum were used as negative or as positive controls.

The specific antibody producing hybridoma colonies were transferred into24-well plates for cell propagation and were tested again. Thehybridomas repeated positive for human MCP-1 N1pE-38 and negative forhuman MCP-1 D3-38 were additionally tested by SPR analysis (Biacore3000).

The best clones which were selected from those showing high associationand low dissociation of bound hMCP1 N1pE-38 and additionally showing nobinding to the negative control peptide MCP1 D3-38, were then cloned andrecloned by limiting-dilution technique, characterized and frozen. Forthe isotype characterization the Mouse Monoclonal Antibody Isotyping Kit(Roche) was used.

Two weeks after fusion of splenocytes from a Balb/c mouse immunized withthe human MCP-1 N1pE-9-BSA-conjugate (short time immunisation protocol)44 cell culture supernatants were primarily tested positive for thepeptide human MCP-1 N1pE-38 as well as for IgG and negative for thepeptide human MCP-1 D3-38. 33 out of 44 hybrids were repeated positiveafter transferring in 24-well plates and were also tested by SPRanalysis (Biacore 3000). 8 hybridomas were cloned by limiting dilutionresulting in 18 specific clones from which 8 were recloned. Thereby, twocell lines were established which produced antibodies with strongreactivity to the peptide human MCP-1 N1pE-38 and good bindingcharacteristics as demonstrated by SPR analysis (Biacore 3000). Theresulting antibodies were designated 332-4B8 and 332-4F8 and bothmonoclonals 332-4B8 and 332-4F8 belong to the IgG class with the isotypeIgG1.

The fusion of the spleen cells of the long time immunized Balb/c micewith cells from mouse myeloma cell line SP2/0 resulted in 35 cellculture supernatants primarily tested positive for human MCP-1 N1pE-38as well as for IgG. Out of these 35 primary positive hybridomas 15 wererepeated positive after transferring in 24-well plates and were furthertested by SPR analysis (Biacore 3000). 4 hybridomas were clonedresulting in 8 specific clones from which 4 were recloned. Finally, twocell lines with a reactivity to the peptide human MCP-1 N1pE-38 but nothuman MCP-1 D3-38 were established as demonstrated by SPR analysis(Biacore 3000). These two cell lines 348-1D4 and 348-2C9 belong to theIgG class with the isotype IgG2b.

From each of the above clones 10 mg of Protein-G purified antibody wasproduced and subjected to further characterization experiments. Theproperties of the various MCP-1 N1pE monoclonal antibodies which wereprepared can be taken from the following examples.

Example 2 SPR Analysis (Biacore 3000) of Generated Monoclonal AntibodiesDirected Against MCP-1 N1pE

Protein-G purified monoclonal antibodies 332-4B8, 332-4F8, 348-1D4 and348-2C9 were characterized with regard to their binding characteristicsto human MCP1 N1pE-38 by SPR analysis. These analyses were performed ona Biacore 3000. To this avail, a CM5 chip was coated with approximately100 response units (RU) human MCP1 N1pE-38 peptide on flow cell (Fc)2.Fc 4 was coated with 100 RU human MCP1 D3-38 peptide. Fc1 and Fc3 wereprepared for blank subtraction. Monoclonal antibodies were diluted inrunning buffer HBS-EP (Hepes buffered saline +3 mM EDTA+0.0050 (v/v)surfactant P20, Biacore, Freiburg, DE) at concentrations ranging from 20μg/ml to 1 μg/ml. First, a basal signal was determined with HBS-EP,followed by 180 seconds of application of antibody dilution, todetermine association of antibody to antigen. Then pure HBS-EP wasinjected again for another 180 sec to determine the dissociation rate ofthe corresponding antibody. Finally, the Biacore CM5 chip wasregenerated by a short injection of 0.1M HCl, to remove all residingantibody.

Blank signals from Fc 1 and Fc 3 were subtracted from signals of Fc 2and Fc 4, respectively.

Results:

All tested monoclonal antibodies failed to associate to human MCP-1D3-38 (data not shown). Regarding association to human MCP-1 N1pE-38,the monoclonal antibodies exhibited different binding characteristics.

The strongest association to human MCP-1 N1pE-38 was demonstrated by332-4B8. At a concentration of 20 μg/ml nearly 2000 RU could bemonitored. Dissociation was almost 0 at a concentration of 5 μg/ml,demonstrating an extremely strong binding of 332-4B8 to its antigen(FIG. 1A).

Also, monoclonal antibody 332-4F8 showed a very stable binding to humanMCP-1 N1pE-38. However, this antibody clone achieved only about 150 RUwithin 180 seconds association time. On the other hand, no dissociationoccurred at all concentrations tested (FIG. 1B).

Monoclonal antibody clones 348-2C9 and 348-1D4 exhibited almost equalbinding characteristics. At 20 μg/ml an association signal ofapproximately 500 RU was monitored for both clones. Dissociation ratedecreased with lower antibody concentrations. Although dissociation wasalso observable at 1 μg/ml of antibody, the measured signals stayed wellabove basal line. (FIG. 1C+FIG. 1D).

Taken together the results provide evidence that all monoclonalantibodies tested are able to interact with their corresponding antigenMCP-1 N1pE.

Example 3 Dot Blot Analysis of Generated Monoclonal Antibodies DirectedAgainst MCP-1 N1pE

Next it was tested, whether the differences in binding kinetics, asdetermined by SPR analysis are also evident in an experimental situationwhere an employed antibody is allowed to interact with its antigen for aprolonged period of time.

A simple DotBlot protocol was accomplished to obtain a general ideaabout the sensitivity of MCP-1 N1pE antibody clones toward therespective native peptide. Human MCP-1 N1pE-38 and human MCP-1 D3-38peptides in descending concentrations (1000 ng-20 ng) were spotted ontosmall pieces of nitrocellulose membranes. For analysis, membranes wereblocked for two hours with TBST-M (=TBST (Tris buffered saline +0.05%Tween-20)+50 skimmed milk) at room temperature with gentle shaking.Membranes were incubated over night at 4° C. on a rocking platform withthe individual MCP-1 N1pE antibody clones diluted to 1 μg/ml in equalvolumes of TBST-M. Secondary anti-mouse antibody conjugated withalkaline phosphatase was used for signal detection, following standardprocedures.

Results:

As seen in FIG. 2A-2D, all antibody clones tested revealed almost equalresults in the Dot Blot analysis. MCP-1 N1pE-38 peptide concentrationsdown to 20 ng were clearly detected by protein-G purified monoclonalantibodies 332-4B8, 332-4F8, 348-1D4 and 348-2C9. None of the antibodyclones generated cross reactivity with MCP-1 D3-38.

Example 4 PepSpot Analysis of Generated Monoclonal Antibodies DirectedAgainst MCP-1 N1pE

To determine specificity and selectivity of MCP-1 N1pE antibody clonesin more detail, PepSpot analysis was performed.

Corresponding PepSpot membranes were prepared by JPT PeptideTechnologies GmbH, Berlin (JPT). On these membranes, peptides with theindicated amino acid sequences (see FIG. 3; Z represents pE) wereimmobilized at a concentration of 1 μg/spot.

For analysis, membranes were blocked for two hours with TBST-M (=TBST+5%skimmed milk) at room temperature with gentle shaking. Membranes wereincubated over night at 4° C. on a rocking platform with the individualMCP-1 N1pE antibody clones diluted to 1 μg/ml in equal volumes ofTBST-M. Secondary anti-mouse antibody conjugated with alkalinephosphatase was used for signal detection, following standardprocedures.

Results:

As seen in FIG. 3 all four antibody clones tested are highly selectivefor MCP-1 N1pE peptides. Strong signals were obtained on spotscontaining MCP-1 N1pE peptides starting with the first 4 amino acids ofMCP-1 N1pE (underlined sequences).

In addition, spots with peptides starting with the first three aminoacids of MCP-1 N1pE (spots 10, 13 and 14) were clearly recognized by allfour antibody clones. Antibodies failed to recognize a peptide startingwith only two of the amino terminal amino acids of MCP-1 N1pE (spot 12).Antibodies also failed to recognize peptide spots starting with aminoacids different than pE (Z). In case of spot 3, spontaneous formation ofpE from Q cannot be excluded. Therefore, signals obtained with thispeptide most likely reflect binding of antibody to spontaneously formedpE.

Taken together the results demonstrate that all four MCP-1 N1pE antibodyclones require the first 3-4 amino acids of MCP-1 N1pE for binding tothe corresponding antigen.

Example 5 Examination of Cross Reactivity to Other Peptides PossessingN1pE Residues by SPR Analysis

In order to determine selectivity of the anti MCP-1 N1pE antibodies,cross reactivity to other human Peptides, possessing a N-terminal pEresidue was analyzed by surface plasmon resonance.

Therefore, the following peptides or there N-terminal regions wereimmobilized on the surface of CM5-Chips: MCP-1, MCP-2, big gastrin,gonadoliberin, neurotensin, orexin A, fibronectin, collagen 1 and TRH.As positive control also the binding to MCP-1 N1pE-38 was analyzed. Themonoclonal antibodies 332-4B8, 332-4F8, 348-1D4 and 348-2C9 were dilutedin HBS-EP (Biacore) down to 25 μg/ml. Cross reactivity was observedusing a Biacore 3000 with several CM5-Chips, on which the respectivepeptides were immobilized. The system was run with 20 μl/min. Measuredbulk effects and unspecific reactions to the chip surface were correctedby subtraction of the signal of flow cell 2, 3 or 4, at which the testedpeptides were immobilized, with the empty flow cell 1. The association(9 min) was obtained by injection of 180 of the antibody clones. Thedissociation was observed over 9 min. Remaining antibody molecules wereremoved by injection of 5 μl 0.1 M HCL. For every interaction of theantibody with the different peptides the association and dissociationwas recorded. Cross reactivity was determined by evaluation of theassociation phase concerning rate and signal at the end.

Table 2 shows, that the monoclonal antibodies are specific for the MCP-1N1pE epitope. No cross reactivity with the analysed peptides wasobserved.

TABLE 2 Investigation of cross reactivity of the monoclonal antibodies332-4B8, 332-4F8, 348-1D4 and 348-2C9 to several human peptides coveringa N-terminal pGlu (pE) residue by SPR analysis. pGlu Peptides % crossreactivity MCP-1 (1-38) 100 MCP-2 <1 MCP-1 (2-38) <1 MCP-1 (3-38 <1Abeta pE3-40 <1 Big Gastrin <1 Gonadoliberin <1 Neurotensin <1 Orexin A<1 Fibronectin <1 Collagen 1 <1 TRH <1

Example 6 Determining K_(D) Values of Monoclonal Antibodies DirectedAgainst MCP-1 N1pE

In order to analyze binding kinetics of the MCP-1 N1pE antibody clonesto hMCP-1 N1pE-38, the association constant K, dissociation constantK_(D), reaction enthalpy ΔH as well as reaction entropy ΔS have beendetermined.

The binding affinities of the anti MCP1 N1pE antibodies 348-1D4 and332-4B8 to the antigen hMCP-1 N1pE-38 were determined using VP-ITCmicrocalorimeter (MicroCal). Both antibody clones as well as the hMCP-1N1pE-38 peptide were dialyzed against 150 mM NaCl, 25 mM Na₂HPO₄, 25 mMKH₂PO₄, 2 mM EDTA pH 7.4 overnight at 4° C. to ensure the same bufferconditions and avoid background heat by protonation events. Afterwardsthe concentration of the antibodies and the peptide was calculated fromabsorbance at 280 nm and the respective extinction coefficient. For thetitration experiment with clone 348-1D4, Antibody and hMCP-1 N1pE-38were used at concentrations of 4.38 μM and 147 μM, respectively. For thetitration experiment with clone 332-4B8, Antibody and hMCP-1 N1pE-38were used at concentrations of 1.86 μM and 64.3 μM, respectively. Thebinding heat was recorded at 20° C. by titration of 29 injections of 10μl of antigen into the antibody solution. In order to evaluate the heatdevelopment originated by the dilution of the hMCP-1 N1pE-38 peptide,this value was determined by titration into the dialysis buffer usingdefined conditions and instrument setup. Plotting of data occurred byMicroCal ORIGIN software. The calculated binding heat was corrected bythe heat originated by dilution of the antigen. The resulting curve wasfitted by the “One Set of Sites” binding model. With this model, thestoichiometry, association constant, reaction enthalpy and reactionentropy can be calculated.

Results:

FIG. 16 shows the resulting fitting curves and the values calculated forstoichiometry, association constant, reaction enthalpy and reactionentropy. In addition, Table 3 gives an overview about the obtained data.

TABLE 3 Parameter obtained for stoichiometry, association anddissociation constant, reaction enthalpy and reaction entropy aftertitration of the antigen hMCP-1 N1pE-38 to the monoclonal antibodies348-1D4 and 332-4B8. 348-1D4 332-4B8 Stoichiometry (N)   1.98   1.60Association Constant 3.81 × 10⁶ 4.27 × 10⁶ (K) in M⁻¹ Dissociation   2.6× 10⁻⁷  2.34 × 10⁻⁷ Constant (K_(D)) in M Reaction Enthalpy −1.123 ×10⁴  −1.823 × 10⁴  (ΔH) in cal/mol Reaction Entropy −8.20 −31.8 (ΔS) incal/mol*K

Example 7 Detection of Recombinant Human MCP-1 N1pE in an ELISA by UsingMonoclonal Antibodies Directed Against MCP-1 N1pE

With the techniques presented so far, selective detection of MCP-1 N1pEcould be clearly demonstrated. Therefore the presented antibody cloneswere also tested for their applicability in tools for potentialdiagnostic implications, like ELISA.

Consequently, an ELISA protocol was accomplished that allowed detectionof recombinant hMCP-1 N1pE.

To capture human MCP-1, commercially available polyclonal antiserum(goat anti-hMCP1-AF (R&D Systems, Minneapolis, USA) as capture antibodywhich specifically binds human MCP-1 was immobilized in polystyrene96-well microtitre plates. Unbound capture antibody was washed off theplate. After a blocking step, recombinant hMCP-1 N1pE diluted inblocking buffer was added to the wells. After an incubation period of 2hours at room temperature, plates were washed at least three times withTBS-T. For detection, MCP-1 N1pE antibody clones (332-4B8, 348-1D4,348-2C9, respectively) together with HRP-conjugated anti mouse antibodywere diluted in blocking buffer, added to the micro titre plate andincubated for 1 hour at room temperature. Following several washes withTBS-T a colour reaction with commercially available HRP substrate TMB(SureBlue Reserve TMB Microwell Peroxidase Substrate (1-component) (KPL,Gaithersburg, USA) was performed (30 minutes incubation at roomtemperature in the dark) and subsequently stopped by the addition of1.2N H₂SO₄. Absorption was determined by a Tecan Sunrise plate reader.

Results:

The anti MCP-1 antibodies 332-4B8, 348-1D4 and 348-2C9 are able todetect recombinant human MCP-1 in a concentration dependent manner.Thereby, the antibody clones 348-2C9 and 348-1D4 turned out to be muchmore sensitive in comparison to 332-4B8 (FIG. 4).

Example 8 Detection of MCP-1 N1pE by ELISA in Human Serum UsingMonoclonal Antibodies Directed Against MCP-1 N1pE

Since recombinant hMCP-1 N1pE can be quantitatively detected in an ELISAby using the monoclonal anti MCP-1 N1pE antibodies of the presentinvention, the detection of native hMCP-1 N1pE in human serum wastested.

The ELISA protocol corresponds to Example 7, except the usage of FBS,0.05% Tween, 10% FBS for blocking and dilution steps.

Results:

All MCP-1 N1pE antibody clones tested, generated very strong signals inthe established ELISA set up. Signals decreased with the dilution factorof the serum sample (FIG. 5; Table 4).

TABLE 4 Detection of human MCP-1 N1pE from human serum by monoclonalantibodies 322-4B8, 348-1D4 and 348-2C9 in ELISA Dilution of Human Serum1:5 1:10 1:20 MCP-1 N1pE Absorption/SD Absorption/SD Absorption/SDAntibody Clone 2.1/0.054 1.5/0.048 0.9/0.008 332-4B8 2.4/0.012 2.1/0.0481.4/0.033 348-1D4 2.5/0.036 2.2/0.042 1.4/0.034 348-2C9

Although clone 332-4B8 demonstrated more favourable bindingcharacteristics in SPR analysis (Biacore), clones 348-1D4 and 348-2C9gave higher signals in the ELISA. According to the obtained data, allantibody clones tested are well suitable for ELISA applications.

Additionally, these data demonstrate that MCP-1 N1pE is detectable alsoin human serum of healthy individuals.

Example 9 Spike and Recovery of hMCP-1 N1pE in Human Serum

Spike and Recovery experiments were performed in order to validate thequantitative detection of hMCP1 N1pE in human serum.

The ELISA protocol corresponds to Example 8, for detection of hMCP-1N1pE the antibody 348-2C9 was used. For validation of Spike and Recoveryvarious levels of recombinant hMCP-1 N1pE were spiked in human serum.Recovery was calculated by subtracting the hMCP-1 N1pE value measured inthe unspiked serum sample from the spiked samples.

Results:

Table 5 shows Spike and Recovery data in human serum obtained with the348-2C9 antibody. A Recovery of the spiked hMCP-1 N1pE peptides of66%-79.4% was found.

TABLE 5 Spike and Recovery of hMCP-1 N1pE in human serum. Expected SpikeLevel Observed Spike Level Observed Spike Level of hMCP1 N1pE of hMCP1N1pE of hMCP1 N1pE [ng/ml] [ng/ml] in % 6 4.76 79.37 3 2.09 69.80 1.51.05 69.81 0.75 0.50 66.00 0.38 0.26 69.89 This table shows the expectedspike level in comparison to observed hMCP-1 N1pE concentrations.

These data confirm, that the monoclonal antibody 348-2C9 can be used forthe quantitative detection of hMCP1 N1pE in human serum.

Example 10 Detection of Human MCP-1 N1pE in Cell Culture Supernatants ofStimulated NHDF Cells by ELISA

Following an inflammatory stimulus, the expression of hMCP-1 is enhancedin Human Normal Dermal Fibroblasts (NHDF). Hence, it can be assumed thatalso MCP-1 N1pE level are elevated. If this holds true, the amount ofMCP-1 N1pE should increase after application of Oncostatin M (OSM) andInterleukin 1β (IL1β) to NHDF.

To prove this, cell culture supernatants of OSM and IL1β stimulated NHDFwere subjected to an ELISA analysis as described in Example 7. Theantibody 348-1D4 was used for detection of hMCP1 N1pE. NHDF have beenstimulated over 14 days and analyzed at different time points in orderto examine time dependency of hMCP-1 N1pE secretion.

Results:

Following the inflammatory stimulus of OSM and IL1β application, theamount of hMCP1 N1pE increases in a time dependent manner (FIG. 6).These data show, that hMCP-1 N1pE can also be quantitatively detected incell culture supernatant of NHDF.

Example 11 Detection of Human MCP-1 N1pE in Cell Culture Supernatant ofLPS Stimulated Human Acute Monocytic Leukemia Cell Line (THP1) in thePresence of QC Inhibitor QCI

As demonstrated in Examples 8-10, native hMCP-1 N1pE can bequantitatively detected in human serum as well as in cell culturesupernatants by ELISA, using the monoclonal anti hMCP-1 N1pE antibodiesof the present invention. As glutaminyl cyclase (QC) is a prerequisitefor MCP-1 N1pE formation on cellular level, inhibition of QC shouldconsequently result in decreased MCP-1 N1pE level.

To prove this, the Human Acute Monocytic Leukemia Cell Line (THP1) wasstimulated 24 h with LPS in absence or presence of increasingconcentrations of the QC inhibitor QCI. Cell culture supernatants weresubjected to ELISA analysis as described in Example 7. The antibody348-1D4 was used for detection of hMCP1 N1pE.

Results:

FIG. 7 shows, that the amount of hMCP-1 N1pE decreases with increasingconcentrations of QC inhibitor in cell culture supernatant of THP1cells.

Example 12 Detection of Recombinant Mouse MCP-1 N1pE in an ELISA byUsing Monoclonal Antibodies Directed Against MCP-1 N1pE

Example 7 shows the concentration dependent detection of recombinanthuman MCP-1 N1pE by the anti MCP-1 antibodies 332-4B8, 348-1D4 and348-2C9. Since the four N-terminal amino acids of mouse and human MCP-1are homologue, the quantitative detection of recombinant mouse MCP-1 wasfurther analysed.

Consequently, an ELISA protocol was accomplished that allowed thedetection of recombinant mouse MCP-1 N1pE.

To capture mouse MCP-1, commercially available polyclonal antiserum(rabbit anti mJE (Peprotech, Rocky Hill, USA) as capture antibody whichspecifically binds mouse MCP-1 was immobilized in polystyrene 96-wellmicrotitre plates. Unbound capture antibody was washed off the plate.After a blocking step, recombinant mMCP-1 N1pE diluted in blockingbuffer was added to the wells. After an incubation period of 2 hours atroom temperature, plates were washed at least three times with TBS-T.For detection, MCP-1 N1pE antibody clones (332-4B8, 348-1D4, 348-2C9,respectively) together with HRP-conjugated anti mouse antibody werediluted in blocking buffer, added to the micro titre plate and incubatedfor 1 hour at room temperature. Following several washing steps withTBS-T a colour reaction with commercially available HRP substrate TMB(SureBlue Reserve TMB Microwell Peroxidase Substrate (1-component) (KPL,Gaithersburg, USA) was performed (30 minutes incubation at roomtemperature in the dark) and subsequently stopped by the addition of1.2N H₂SO₄. Absorption was determined by a Tecan Sunrise plate reader.

Results:

The anti MCP-1 antibodies 332-4B8, 348-1D4 and 348-2C9 are able todetect recombinant mouse MCP-1 in a concentration dependent manner.Similar to the results obtained with human hMCP-1, the antibody clones348-2C9 and 348-1D4 turned out to be much more sensitive in comparisonto 332-4B8 (FIG. 8).

Example 13 Detection of Mouse MCP-1 N1pE in Cell Culture Supernatants ofa Stimulated Murine Macrophage Cell Line RAW 264.7 by ELISA

Following an inflammatory stimulus, the expression of mMCP-1 is enhancedin RAW 264.7 cells. Hence, it can be assumed that also MCP-1 N1pE levelare elevated. If this holds true, the amount of mMCP-1 N1pE shouldincrease after application of LPS.

To prove this, cell culture supernatants of LPS stimulated RAW 264.7were subjected to an ELISA analysis as described in Example 12. Theantibody 348-2C9 was used for detection of mMCP1 N1pE. RAW 264.7 cellshave been stimulated for 24 h with 10 ng LPS.

Results:

Following the inflammatory stimulus of LPS application, the amount ofmMCP1 N1pE increases tremendously (FIG. 9). These data show that mMCP-1N1pE can also be quantitatively detected in cell culture supernatant ofRAW 264.7 cells.

Example 14 Detection of Murine MCP-1 N1pE by ELISA in Cell CultureSupernatants of LPS Stimulated Murine Macrophage Cell Line RAW 264.7 inthe Presence of QC Inhibitor QCI Using Monoclonal Antibody 348-2C9

As demonstrated in Example 13, the mMCP-1 N1pE level increasesrespectably after an inflammatory stimulus like LPS. It could further beshown in Example 11, that the anti MCP-1 N1pE antibodies can be used todemonstrate that the human MCP-1 N1pE level decreases with increasingconcentrations of QCI. It has now further been examined if this effectcan also be detected in the mouse Macrophage cell line RAW 264.7.

The mouse macrophage cell line RAW 264.7 was stimulated with LPS in theabsence or presence of increasing concentrations of the QC inhibitorQCI. Cell culture supernatants were subjected to ELISA analysis asdescribed in Example 12. For detection the antibody 348-2C9 was used.

Results:

As postulated before, mMCP-1 N1pE level drops in the presence of the QCinhibitor QCI in LPS stimulated mouse macrophages. The decrease of thesignal is strictly dependent on the concentration of QCI (see FIG. 10).

Example 15 Detection of Murine MCP-1 N1pE in Serum of Healthy MiceVersus LPS Treated Mice by ELISA

Examples 12-14 show the quantitative detection of recombinant mMCP1 N1pEas well as native mMCP1 N1pE in cell culture supernatant by ELISA. Aspresented in Examples 8-9, the anti MCP-1 N1pE antibodies can also beused for the detection of MCP-1 N1pE in human serum. Further, the levelof MCP-1 N1pE is now determined in mouse serum.

The ELISA protocol corresponds to Example 12, except the usage of FBS,0.05% Tween, 10% FBS for blocking and dilution steps and the usage ofthe antibody 348-2C9 for detection.

Results:

The mMCP-1 N1pE level in mouse serum increases by LPS stimulation,depending on the time period of stimulation from 400 pg/ml up to 900ng/ml (FIG. 11). This experiment shows that the antibody 348-2C9 canalso be used for the quantitative detection of mMCP-1 N1pE in murineserum.

Example 16 Examination of Dilution Linearity for the Detection of mMCP-1N1pE in Murine Peritoneal Lavage Fluid by ELISA

In order to examine the applicability of the anti MCP-1 N1pE antibodiesin the established ELISA, the dilution linearity of Peritoneal LavageSamples from mice treated with Thioglycollate was analyzed.

The ELISA protocol corresponds to Example 12. For detection, theantibody 348-2C9 was used. To determine assay linearity, each sample wasserially diluted with ELISA Blocker to produce values that are withinthe assay range.

Results:

FIG. 12 depicts, that the analysis of different sample dilutions resultsin similar mMCP-1 N1pE levels with deviations of 15% maximum. Thisexperiment demonstrates, that the anti MCP-1 N1pE antibodies can be usedfor the analysis of MCP-1 N1pE level in mouse peritoneal lavage fluid.

Example 17 Usage of Anti MCP-1 N1pE Antibodies in Western Blot Analysisand Comparison to Data Obtained by ELISA

Examples 3-4 reveal, that the antibodies 332-4F8, 332-4B8, 348-1D4 and348-2C9 recognize at least the first 4 amino acids of MCP-1 N1pE in DotBlot and PepSpot analysis. In this experiment, it should be testedwhether the antibodies can be used for the detection of native mouseMCP-1 N1pE in cell culture supernatant of RAW 264.7. Furthermore, itshould be tested, whether the obtained Western Blot data can confirm thedata obtained by ELISA.

For Western Blot analysis, cell culture supernatants of RAW 264.7 cellswere subjected to SDS-gelelektrophoresis. Separated proteins weretransferred electrically to a nitrocellulose membrane. After blocking ofthe membrane for two hours with TBST-M at room temperature, antibodyincubation occurred over night at 4° C. with the anti MCP-1 N1pE clone332-4B8 and an antibody recognizing total MCP-1 (goat anti MCP-1, R&DSystems) diluted to 1 μg/ml in TBST-M. Secondary goat anti mouseantibody conjugated with horseradish peroxidase was used for signaldetection, following standard procedures.

The ELISA protocol corresponds to Example 14.

Results:

As shown in FIG. 13B, there is no change in the Western Blot signalintensity generated by the antibody goat anti Mouse MCP-1 for thedetection of total mMCP-1. However, the Western Blot signal of mMCP-1N1pE is concentration dependent (FIG. 13A) and correlates with thecorresponding ELISA data (FIG. 13C), showing the amount of mMCP-1 N1pE.These data show on the one hand that the anti MCP-1 N1pE antibody332-4B8 can be used for Western Blot analysis. Furthermore, thecorrectness of the ELISA data by Western Blot analysis was confirmed.

Example 18 Detection of Recombinant Rat MCP-1 N1pE in an ELISA by UsingMonoclonal Antibodies Directed Against MCP-1 N1pE

Example 12 shows the concentration dependent detection of recombinantmouse MCP-1 N1pE by the anti MCP-1 antibodies 332-4B8, 348-1D4 and348-2C9. The N-terminal sequences of mouse and rat MCP-1 are homologue.Therefore, the quantitative detection of recombinant rat MCP-1 wasanalysed.

Consequently, an ELISA protocol was accomplished that allowed thedetection of recombinant rat MCP-1 N1pE.

To capture rat MCP-1, commercially available polyclonal antiserum(rabbit polyclonal to MCP-1 [LS-054182/13136], LifeSpan Biosciences,Seattle, USA) as capture antibody which specifically binds rat MCP-1 wasdiluted with PBS to 250 ng/ml and immobilized in polystyrene 96-wellmicrotitre plates. Unbound capture antibody was washed off the plate.After a blocking step, recombinant rMCP-1 N1pE diluted in PBS, 0.050Tween, 10% FBS was added to the wells. After an incubation period of 2hours at room temperature, plates were washed at least three times withTBS-T. For detection, the MCP-1 N1pE antibody clone 348-2C9 togetherwith HRP-conjugated anti mouse antibody were diluted in PBS, 0.05%Tween, 10% FBS, added to the micro titre plate and incubated for 1 hourat 4° C. Following several washes with TBS-T a colour reaction withcommercially available HRP substrate TMB (SureBlue Reserve TMB MicrowellPeroxidase Substrate (1-component) (KPL, Gaithersburg, USA) wasperformed (30 minutes incubation at room temperature in the dark) andsubsequently stopped by the addition of 1,2N H₂SO₄. Absorption wasdetermined by a Tecan Sunrise plate reader.

Results:

The anti MCP-1 antibody 348-2C9 is able to detect recombinant rat MCP-1in a concentration dependent manner in an ELISA (FIG. 14).

Example 19 Spike and Recovery of rMCP-1 N1pE in Rat Serum

Example 18 shows the quantitative detection of recombinant rat MCP-1 bythe antibody 348-2C9 in an ELISA. In order to proof whether rMCP-1 N1pEcan also be detected in rat serum and to validate this ELISA method,Spike and Recovery experiments were performed.

The ELISA protocol corresponds to Example 18, for validation of Spikeand Recovery various levels of recombinant rat MCP-1 N1pE were spiked inserum of LPS treated rats. Recovery was calculated by subtracting therMCP-1 N1pE value measured in the unspiked serum sample from the spikedsamples.

Results:

Table 6 shows Spike and Recovery data in rat serum obtained with the348-2C9 antibody. A Recovery of the spiked rMCP-1 N1pE peptides of65.50-96.2% was found.

TABLE 6 Spike and Recovery of rMCP-1 N1pE in serum of LPS stimulatedrats. Expected Spike Level Observed Spike Level Observed Spike Level ofrMCP1 N1pE of rMCP1 N1pE of rMCP1 N1pE [ng/ml] [ng/ml] in % 2000 148574.23% 1000 962 96.20% 500 327 65.49% 250 212 84.83% This table showsthe expected spike level in comparison to observed rMCP-1 N1pEconcentrations.

These data confirm, that the monoclonal antibody 348-2C9 can be used forthe quantitative detection of rat MCP1 N1pE in rat serum.

Example 20 Sequencing Antibody Variable Regions Cultivation of HybridomaCells:

Hybridoma cells were grown in D-MEM (+L-Glutamin, +Na-Pyruvat, 4.5 g/lGlucose, Gibco) with the addition of 15% FBS, 1% MEM-NEA (non essentialamino acids, Gibco), 50 μg/ml Gentamycin (Gibco) and 50 μMβ-mercaptoethanol at 37° C. and 5% CO₂. Subcultivation occurred after3-4 days depending on cell density. Cells were seeded in a concentrationof 0.5×10⁶ cells/ml, splitting occurred at a cell density of 2-5×10⁶cells/ml.

cDNA Synthesis and Reverse Transcription:

Total RNA was isolated from 2×10⁶ cells according to the manual of theNucleospinRNA Isolation Kit (Macherey-Nagel). 100 ng RNA were appliedfor cDNA synthesis by using Oligo (dT)₁₅ primer (Promega) andSuperScript III Reverse Transcriptase (Invitrogen).

PCR-Amplification of Heavy and Light Chain Variable Regions:

Heavy chain variable regions were amplified from the template cDNA byusing Phusion™ High-Fidelity DNA Polymerase (NEW ENGLAND BioLabs) withthe primer MHCG1 (in case of clone 5-5-6 and 6-1-6) and MHCG2b (clone17-4-3 and 24-2-3) in combination with primers MHV1-12. Foramplification of light chain variable regions the primer MKC incombination with the primers MKV1-MKV11 were used.

Cloning of PCR Products in pJET1.2:

Heavy and light chain variable regions, amplified by PCR, were clonedinto pJET1.2/blunt vector according to the protocol of CloneJET™ PCRCloning Kit (Fermentas). Sequencing occurred with pJET1.2 sequencingprimers. The primer sequences are shown in Table 7.

TABLE 7 Primer sequences SEQ Primer Sequence ID NO. MKV1ATGAAGTTGCCTGTTAGGCTGTTGGTGCTG  7 MKV2 ATGGAGWCAGACACACTCCTGYTATGGGTG  8MKV3 ATGAGTGTGCTCACTCAGGTCCTGGSGTTG  9 MKV4ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG 10 MKV5 ATGGATTTWCAGGTGCAGATTWTCAGCTTC11 MKVG ATGAGGTKCYYTGYTSAGYTYCTGRGG 12 MKV7ATGGGCWTCAAGATGGAGTCACAKWYYCWGG 13 MKV8 ATGTGGGGAYCTKTTTYCMMTTTTTCAATTG14 MKV9 ATGGTRTCCWCASCTCAGTTCCTTG 15 MKV10 ATGTATATATGTTTGTTGTCTATTTCT16 MKV11 ATGGAAGCCCCAGCTCAGCTTCTCTTCC 17 MKC ACTGGATGGTGGGAAGATGG 18MHV1 ATGAAATGCAGCTGGGGCATSTTCTTC 19 MHV2 ATGGGATGGAGCTRTATCATSYTCTT 20MHV3 ATGAAGWTGTGGTTAAACTGGGTTTTT 21 MHV4 ATGRACTTTGGGYTCAGCTTGRTTT 22MHV5 ATGGACTCCAGGCTCAATTTAGTTTTCCTT 23 MHV6 ATGGCTTGTCYTRGSGCTRCTCTTCTGC24 MHV7 ATGGRATGGAGCKGGRTCTTTMTCTT 25 MHV8 ATGAGAGTGCTGATTCTTTTGTG 26MHV9 ATGGMTTGGGTGTGGAMCTTGCTATTCCTG 27 MHV10 ATGGGCAGACTTACATTCTCATTCCTG28 MHV11 ATGGATTTTGGGCTGATTTTTTTTATTG 29 MHV12ATGATGGTGTTAAGTCTTCTGTACCTG 30 MHCG1 CAGTGGATAGACAGATGGGGG 31 MHCG2bCAGTGGATAGACTGATGGGGG 32

Results:

The following sequences were identified:

Clone 1D4

Light chain variable part, nucleotide sequence (SEQ ID NO: 33)ATGGAGTCACAGTTTCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGTGATGTTGTGATGACCCAGACTCCCCTCAGTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGCTGGAAAGACATATTTGAGTTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCTGGCAAGGTACACATTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT Light chain variable part, proteinsequence (SEQ ID NO: 34)MESQFLFLLVLWIRETNGDVVMTQTPLSLSVTIGQPASISCKSSQSLLDSAGKTYLSWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIKRADAAPTVSIFPPSS Heavy chain variable part,nucleotide sequence (SEQ ID NO: 35)ATGGAATGGAGCGGGGTCTTTCTCTTCCTCTTGTCAGGAACTGCAGGTGTCCACTCTGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTACATGGACTGGGTGAAGCAGAGCCATGGAGAAAGCTTTGAGTGCATTGGACGTGTTAATCCTTACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCAACAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGCTCGGTAGTAGCTACCGCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACACCCCCATCAGTCT Heavy chain variable part, protein sequence(SEQ ID NO: 36) MEWSGVFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMDWVKQSHGESFECIGRVNPYNGGTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARLGSSYRWGQGTTLTVSSAKTTPPSV

Clone 2C9

Light chain variable part, nucleotide sequence (SEQ ID NO: 37)ATGGTGTCCTCAGCTCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGTGATGTTGTGATGACCCAGACTCCCCTCAGTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGCTGGAAAGACATATTTGAGTTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCTGGCAAGGTACACATTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT Light chain variable part,protein sequence (SEQ ID NO: 38)MVSSAQFLFLLVLWIRETNGDVVMTQTPLSLSVTIGQPASISCKSSQSLLDSAGKTYLSWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIKRADAAPTVSIFPPSS Heavy chain variablepart, nucleotide sequence (SEQ ID NO: 39)ATGGAATGGAGCGGGATCTTTATCTTCCTCTTGTCAGGAACTGCAGGTGTCCACTCTGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTACATGGACTGGGTGAAGCAGAGCCATGGAGAAAGCTTTGAGTGCATTGGACGTGTTAATCCTTACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCAACAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGCTCGGTAGTAGCTACCGCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACACCCCCATCAGTCTATCCACTG Heavy chain variable part, proteinsequence (SEQ ID NO: 40)MEWSGIFTFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMDWVKQSHGESFECIGRVNPYNGGTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARLGSSYRWGQGTTLTVSSAKTTPPSVYPL

Clone 4B8

Light chain variable part, nucleotide sequence (SEQ ID NO: 41)ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAAATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTTCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT Light chain variable part,protein sequence (SEQ ID NO: 42)MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCKSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVFNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRADAAPTVSIFPPSS Heavy chain variablepart, nucleotide sequence (SEQ ID NO: 43)ATGGGATGGAGCGGGGTCTTTATTTTAATCCTGTCAGTAACTACAGGTGTCCACTCTGAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGAGAAGCCTGGCGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACAACATGAACTGGGTGAAGCAGAACAATGGAAAGAGCCTTGAGTGGATTGGAAATATTACTCCTTACTATGGTAGTACTAGCTACAACCAGAAGTTCAAGGGCAGGGTCACATTGACTGTGGACAAATCCTCCAGCACAGCCTACATGCAGCTCAAGAGCCTGACATCTGAGGACTCTGCAGTCTATTTCTGCCTCCTATGGTTACGACGGGGGGACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTG Heavy chain variablepart, protein sequence (SEQ ID NO: 44)MGWSGVFILILSVTTGVHSEVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNITPYYGSTSYNQKFKGRVTLTVDKSSSTAYMQLKSLTSEDSAVYFCLLWLRRGDYAMDYWGQGTSVTVSSAKTTPPSVYPL

Example 21 Application of MCP-1 N1pE Antibody Clones forImmunohistochemistry

With the antibodies of the present invention, MCP-1 N1pE was stained inbrain sections of rats after microinjection of Aβ(3-49), LPS or NaCl.The stained brain sections are shown in FIG. 15. FIG. 15 shows that theantibodies 332-4B8, 348-1D4 and 348-2C9 of the present invention aresuitable for immunohistochemistry. The antibodies specifically detectMCP-1 N1pE in brain of rats.

Deposits

Monoclonal antibodies specifically recognizing MCP-1 N1pE, weregenerated. Currently all corresponding monoclonal antibodies expressinghybridoma cell clones 348/1D4, 348/2C9, 332/4B8 and 332/4F8 have beendeposited in accordance with the Budapest Treaty and are available atthe Deutsche Sammlung far Mikroorganismen and Zellkulturen (DSMZ)(German Collection of Microorganisms and Cell Cultures) GmbH,Inhoffenstrasse 7B, 38124 Braunschweig, Germany, with a deposit date ofMay 6, 2008, and with the respective deposit numbers

DSM ACC 2905 (Hybridoma cell clone 348/1D4) DSM ACC 2906 (Hybridoma cellclone 348/2C9) DSM ACC 2907 (Hybridoma cell clone 332/4B8) and DSM ACC2908 (Hybridoma cell clone 332/4F8).

Specificity of those antibodies for their respective target sequencescould be confirmed. For MCP-1 N1pE, high affinity antibody clones couldbe identified that should give strong signals in an ELISA set up with anexpected detection limit in the low pg range.

1. An antibody, or a functionally equivalent antibody or functionalparts thereof, which selectively binds to MCP-1 N1pE.
 2. The antibody ofclaim 1, wherein said antibody is a monoclonal antibody.
 3. The antibodyof claim 1, wherein said antibody is a chimeric antibody.
 4. Theantibody according to claim 1, wherein a variable part of a light chainof said antibody has a nucleotide sequence selected from SEQ ID NOs: 33,37 and 41, or an amino acid sequence selected from SEQ ID NOs: 34, 38and
 42. 5. The antibody to claim 1, wherein a variable part of a heavychain of said antibody has a nucleotide sequence selected from SEQ IDNOs: 35, 39 and 43, or an amino acid sequence selected from SEQ ID NOs:36, 40 and
 44. 6. The antibody to claim 1, wherein the variable part ofthe light chain of said antibody has the nucleotide sequence of SEQ IDNO: 33 or the amino acid sequence of SEQ ID NO: 34, and wherein thevariable part of the heavy chain of said antibody has the nucleotidesequence of SEQ ID NO: 35, or the amino acid sequence of SEQ ID NO: 36.7. The antibody according to claim 1, wherein a variable part of a lightchain of said antibody has a nucleotide sequence of SEQ ID NO: 37 or theamino acid sequence of SEQ ID NO: 38, and wherein a variable part of aheavy chain of said antibody has a nucleotide sequence of SEQ ID NO: 39,or an amino acid sequence of SEQ ID NO:
 40. 8. The antibody according toclaim 1, wherein a variable part of a light chain of said antibody has anucleotide sequence of SEQ ID NO: 41 or an amino acid sequence of SEQ IDNO: 42, and wherein a variable part of a heavy chain of said antibodyhas a nucleotide sequence of SEQ ID NO: 43, or an amino acid sequence ofSEQ ID NO:
 44. 9. The antibody according to claim 1, which is producedby hybridoma cells, wherein said hybridoma cells are selected from thegroup consisting of hybridoma cells deposited with the Deutsche Sammlungfür Mikroorganismen and Zellkulturen GmbH, DSMZ as DSM ACC 2905, DSM ACC2906, DSM ACC 2907 and DSM ACC
 2908. 10. The antibody according to claim1, which antibody comprises a light chain variable region comprising anucleic acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selectedfrom SEQ ID NOs: 33, 37 or
 41. 11. The antibody according to claim 1,which antibody comprises a heavy chain variable region comprising anucleic acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selectedfrom SEQ ID NOs: 35, 39 or 43, or a functional part thereof.
 12. Theantibody according to claim 1, which antibody comprises a light chainvariable domain comprising an amino acid sequence that is 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto a sequence selected from SEQ ID NOs: 34, 38 or
 42. 13. The antibodyaccording to claim 1, which antibody comprises a heavy chain variabledomain comprising an amino acid sequence that is 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to asequence selected from SEQ ID NOs: 36, 40 or
 44. 14. The antibodyaccording to claim 1, wherein: said antibody comprises a variable partof a light chain comprising an amino acid sequence selected from SEQ IDNOs: 34, 38 and 42; or a variable part of a heavy chain comprising anamino acid sequence selected from SEQ ID NOs: 36, 40 and 44; theantibody has been altered by introducing at least one, at least two, orat least 3 or more conservative substitutions into at least one of thesequences of SEQ ID NOs: 34, 36, 38, 40, 42 and 44; and the antibodyessentially maintains its full functionality.
 15. The antibody accordingto claim 1, wherein selectively binding comprises binding to apyroglutamate carrying amino terminus of MCP-1.
 16. The antibodyaccording to claim 1, wherein selectively binding comprises essentiallyno cross-reactivity with an epitope other than a pyroglutamate carryingamino terminus of MCP-1N1pE.
 17. The antibody according to claim 1,wherein said antibody is a humanized antibody.
 18. The antibodyaccording to claim 1, wherein said antibody is an antibody fragment,which retains a high affinity for MCP-1N1pE.
 19. The antibody accordingto claim 1, or a functionally equivalent antibody or functional partsthereof, wherein the antibody specificity for MCP-1 N1pE is sufficientfor detection of MCP-1 N1pE or variants thereof in a mammal.
 20. Theantibody according to claim 19, wherein said mammal is a rat, mouse or ahuman.
 21. The antibody according to claim 1, which is a diabody or asingle chain antibody which retains a high affinity to MCP-1 N1pE. 22.The antibody according to claim 1, wherein complementarity determiningregions of the antibodies comprise a complementarity determining regionof an antibody produced by hybridoma cells selected from the groupconsisting of hybridoma cells deposited with the Deutsche Sammlung fürMikroorganismen und Zellkulturen GmbH, DSMZ as DSM ACC 2905, DSM ACC2906, DSM ACC 2907 and DSM ACC
 2908. 23. The antibody according to claim1, which is labeled.
 24. The antibody according to claim 1, which isimmobilized on a solid phase.
 25. A hybridoma cell line selected fromthe group consisting of DSM ACC 2905, DSM ACC 2906, DSM ACC 2907 and DSMACC 2908, all deposited with the Deutsche Sammlung für Mikroorganismenund Zellkulturen GmbH, DSMZ.
 26. A composition, comprising the antibodyof claim 1, or a functionally equivalent antibody or functional partsthereof.
 27. A pharmaceutical composition comprising the antibody ofclaim 1, or a functionally equivalent antibody or functional partsthereof, in a therapeutically effective amount and, optionally, at leastone of a further biologically active substance, a pharmaceuticallyacceptable carrier, a diluent, or an excipient.
 28. The pharmaceuticalcomposition of claim 27, wherein said further biologically activecompound is selected from the group consisting of neuron-transmissionenhancers, psychotherapeutic drugs, acetylcholine esterase inhibitors,calcium-channel blockers, biogenic amines, benzodiazepinetranquillizers, acetylcholine synthesis, storage or release enhancers,acetylcholine postsynaptic receptor agonists, monoamine oxidase-A or -Binhibitors, N-methyl-D-aspartate glutamate receptor antagonists,non-steroidal anti-inflammatory drugs, antioxidants, serotonergicreceptor antagonists, CCR2 receptor antagonists and MCP-1 antibodies.29. The pharmaceutical composition of claim 27, wherein said furtherbiologically active compound is selected from the group consisting of acompound effective against oxidative stress, anti-apoptotic compounds,metal chelators, inhibitors of DNA repair α-secretase activators, tauproteins, neurotransmitter, β-sheet breakers, attractants for amyloidbeta clearing/depleting cellular components, inhibitors of N-terminaltruncated amyloid beta peptides including pyroglutamated amyloid beta3-42, inhibitors of glutaminyl cyclase, anti-inflammatory molecules,cholinesterase inhibitors (ChEIs), an amyloid or tau modifying drug, andnutritive supplements.
 30. The pharmaceutical composition of claim 27,wherein said further biologically active compound is selected fromPEP-inhibitors, LiCl, inhibitors of DP IV or DP IV-like enzymes;acetylcholinesterase (AChE) inhibitors, PIMT enhancers, inhibitors ofbeta secretases, inhibitors of gamma secretases, inhibitors of neutralendopeptidase, inhibitors of phosphodiesterase-4, TNFalpha inhibitors,muscarinic M1 receptor antagonists, NMDA receptor antagonists, sigma-1receptor inhibitors, histamine H3 antagonists, immunomodulatory agents,immunosuppressive agents or an agent selected from antegren(natalizumab), Neurelan (fampridine-SR), campath (alemtuzumab), IR 208,NBI 5788/MSP 771 (tiplimotide), paclitaxel, Anergix.MS (AG 284), SH636,Differin (CD 271, adapalene), BAY 361677 (interleukin-4),matrix-metalloproteinase-inhibitors (e.g. BB 76163), interferon-tau(trophoblastin) and SAIK-MS; beta-amyloid antibodies, cysteine proteaseinhibitors; MCP-1 antagonists, amyloid protein deposition inhibitors andbeta amyloid synthesis inhibitors.
 31. The pharmaceutical composition ofclaim 29, wherein said further biologically active compound is acholinesterase inhibitor (ChEI).
 32. The pharmaceutical composition ofclaim 31, wherein said cholinesterase inhibitor is selected from thegroup consisting of tacrine, rivastigmine, donepezil, galantamine,niacin and memantine.
 33. The pharmaceutical composition of claim 29,wherein said further biologically active compound is a glutaminylcyclase inhibitor.
 34. The pharmaceutical composition of claim 32,wherein said glutaminyl cyclase inhibitor is1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea-hydrochloride.35. A method for the treatment, prevention or delay of a MCP-1-relatedinflammatory disease or condition comprising: contacting a biologicalsample and an antibody that binds with MCP 1 N1pE, or a functionallyequivalent antibody or functional parts thereof; and detecting specificbinding of the antibody to MCP-1 N1pE in the sample; wherein theMCP-1-related inflammatory disease or condition is selected from thegroup consisting of a a. a neurodegenerative disease; b.neurodegenerative disease selected from the group consisting of mildcognitive impairment (MCI), Alzheimer's disease, neurodegeneration inDown Syndrome, Familial British Dementia, Familial Danish Dementia, andmultiple sclerosis; c. chronic or acute inflammation; d. chronic oracute inflammation selected from the group consisting of rheumatoidarthritis, atherosclerosis, restenosis, and pancreatitis; e. fibrosis;f. fibrosis selected from the group consisting of lung fibrosis, liverfibrosis, and renal fibrosis; g. cancer; h. cancer selected from thegroup consisting of hemangioendothelioma proliferation and gastriccarcinoma; i. metabolic disease j. a metabolic disease selected from thegroup consisting of hypertension; and k. an inflammatory diseaseselected from the group consisting of neuropathic pain, graftrejection/graft failure/graft vasculopathy, HIV infections/AIDS,gestosis, and tuberous sclerosis.
 36. The method of claim 35 wherein theMCP-1-related inflammatory disease or condition is selected from thegroup consisting of atheroschlerosis, rheumatoid arthritis, asthma,delayed hypersensitivity reactions, pancreatitis, Alzheimer's disease,lung fibrosis, renal fibrosis, gestosis, graft rejection, neuropathicpain, AIDS and tumors.
 37. The method of claim 35 wherein theMCP-1-related inflammatory disease or condition is selected from thegroup consisting of atherosclerosis, rheumatoid arthritis, restenosisand pancreatitis.
 38. The method of claim 35 wherein the MCP-1-relatedinflammatory disease or condition is selected from the group consistingof MCI or Alzheimer's disease.
 39. A method for detecting MCP-1 N1pE,comprising: contacting a biological sample and an antibody that bindswith MCP 1 N1pE, or a functionally equivalent antibody or functionalparts thereof; and detecting specific binding of the antibody to MCP-1N1pE in the sample.
 40. The method of claim 39 wherein the method is anin vitro or in situ diagnostic method for the diagnosis of aMCP-1-related disease or condition.
 41. The method of claim 40, whereincontacting the biological sample and the antibody comprises: contactingthe antibody which binds with MCP 1 N1pE, or a functionally equivalentantibody or functional parts thereof, with a sample, or a specific bodypart or body area of a subject suspected to be afflicted with saidcondition or disease.
 42. The method of claim 41, comprising: detectingimmunospecific binding of an antibody which binds with MCP 1 N1pE, or afunctionally equivalent antibody or functional parts thereof, to anMCP-1 N1pE peptide in a sample or in situ, which comprises a. contactingthe antibody and the sample or a specific body part or body areasuspected to contain the MCP-1 peptide; b. allowing the antibody to bindto the MCP-1 N1pE peptide to form an immunological complex; c. detectingformation of the immunological complex; and d. correlating presence orabsence of the immunological complex with presence or absence of MCP-1N1pE peptide in the sample or specific body part or area.
 43. The methodof claim 41, wherein said biological sample is selected from the groupconsisting of a serum, liquor, cerebrospinal fluid (CSF) and synovialfluid sample.
 44. The method of claim 41, wherein said biological sampleis a serum sample.
 45. An isolated polynucleotide encoding a heavy chainvariable region of a monoclonal antibody of claim 5, wherein saidisolated polynucleotide comprises a nucleic acid sequence selected fromSEQ ID Nos: 35, 39 and
 43. 46. An isolated peptide of the light chainvariable region of a monoclonal antibody of claim 4, wherein saidisolated peptide comprises an amino acid sequence selected from SEQ IDNos: 34, 38 and
 42. 47. An isolated peptide of the heavy chain variableregion of a monoclonal antibody of claim 5, wherein said isolatedpeptide comprises an amino acid sequence selected from SEQ ID Nos: 36,40 and
 44. 48. An oligonucleotide selected from the group consisting ofSEQ ID NOs: 7 to 32.